Neutralizing anti-influenza A antibodies and uses thereof

ABSTRACT

The invention relates to antibodies and binding fragments thereof that are capable of binding to influenza A virus hemagglutinin and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza A virus. In one embodiment, an antibody or binding fragment according to the invention is capable of binding to and/or neutralizing one or more influenza A virus group 1 subtypes selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 and variants thereof and one or more influenza A virus group 2 subtype selected from H3, H4, H7, H1, 0, H14 and H15 and variants thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application of InternationalApplication No. PCT/US2014/058652 filed on Oct. 1, 2014, saidInternational Application No. PCT/US2014/058652 claims benefit under 35U.S.C. § 119(e) of the U.S. Provisional Application Nos. 61/885,808,filed Oct. 2, 2013 and 62/002,414, filed May 23, 2014. Each of the abovelisted applications is incorporated by reference herein in its entiretyfor all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled FLUA-100US1_SL, created onMar. 8, 2016, and having a size of 91.9 kilobytes.

FIELD OF THE INVENTION

The invention relates to antibodies that have broad neutralizingactivity against influenza A virus and to uses of such antibodies.

BACKGROUND TO THE INVENTION

Influenza viruses cause annual influenza epidemics and occasionalpandemics, which pose a significant threat to public health worldwide.Seasonal influenza infection is associated with 200,000-500,000 deathseach year, particularly in young children, immunocompromised patientsand the elderly. Mortality rates typically increase further duringseasons with pandemic influenza outbreaks. There remains a significantunmet medical need to develop potent anti-viral therapeutics forpreventing and treating influenza infections, particularly inunder-served populations.

There are three types of influenza viruses, types A, B and C. InfluenzaA viruses can infect a wide variety of birds and mammals, includinghumans, pigs, chickens and ferrets. Influenza A viruses can beclassified into subtypes based on allelic variations in antigenicregions of two genes that encode surface glycoproteins hemagglutinin(HA) and neuraminidase (NA). HA is the receptor-binding and membranefusion glycoprotein, which mediates viral attachment and entry intotarget cells; HA is the primary target of protective humoral immuneresponses. The HA protein is trimeric in structure and is comprised ofthree identical copies of a single polypeptide precursor, HA0, whichupon proteolytic maturation, is cleaved into a pH-dependent, metastableintermediate containing the globular head (HA1) and the stalk region(HA2). The membrane distal “globular head” constitutes the majority ofthe HA1 structure and contains the sialic acid binding pocket for viralentry and major antigenic domains. The membrane proximal “stalk”structure, assembled from HA2 and some HA1 residues, contains the fusionmachinery, which undergoes a conformational change in the low pHenvironment of late endosomes to trigger membrane fusion and penetrationinto cells. The degree of sequence homology between influenza A subtypesis smaller in the HA1 (34%-59% homology between subtypes) than in theHA2 region (51%-80% homology). Neutralizing antibodies elicited byinfluenza virus infection are normally targeted to the variable HA1globular head to prevent viral receptor binding and are usuallystrain-specific. Rarely, broad cross-reactive monoclonal antibodies havebeen identified that target the globular head of HA (Krause J. C. et al.2011 J. Virol. 85; Whittle J. et al., 2011 PNAS 108; Ekiert D C et al.,2012 Nature 489; Lee P S et al., 2012 PNAS 109). In contrast, thestructure of the stalk region is relatively conserved and a handful ofbroadly neutralizing antibodies have recently been identified that bindto HA stalk to prevent the pH-triggered fusion step for viral entry(Ekiert D. C. et al., 2009 Science 324; Sui J. et al., Nat Struct MolBiol 16; Wrammert J et al., 2011 J Exp Med 208; Ekiert D. C et al., 2011Science 333; Corti D et al., 2010 J Clin Invest 120; Throsby M., 2008PLoS One 3). The majority of these stalk reactive neutralizingantibodies are either specific to influenza A group 1 viruses orspecific to group 2 viruses. Very recently, stalk binding antibodieswere isolated that were cross-reactive to both groups 1 and 2 viruses(Corti D. et al., 2011 Science 333; Li G M et al., 2012 PNAS 109 andCyrille D et al., 2012 Science 337; Nakamura G et al., 2013, Cell Host &Microbe 14).

To date, there are no marketed antibodies that broadly neutralize orinhibit all influenza A virus infection or attenuate disease caused byinfluenza A virus. Therefore, there remains a need for new antibodiesthat protect against multiple group 1 and group 2 subtypes of influenzaA virus.

DESCRIPTION OF THE INVENTION

The invention provides an antibody to influenza A virus or a bindingfragment thereof that is capable of binding to influenza A virushemagglutinin and neutralizing at least one group 1 subtype and at least1 group 2 subtype of influenza A virus.

Preferably antibody or binding fragments of the invention are capable ofbinding to influenza A virus hemagglutinin and neutralizing at least 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 influenza A virus group 1 subtype and atleast 1, 2, 3, 4, 5, or 6 influenza A virus group 2 subtypes. Furtherpreferably, antibody or binding fragments of the invention are capableof binding to influenza A virus hemagglutinin and neutralizing at least5 influenza A virus group 1 subtypes and at least 1 or 2 influenza Avirus group 2 subtypes.

The hemagglutinin subtypes of influenza A viruses fall into two majorphylogenetic groupings, identified as group 1, which includes subtypesH1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 and group 2, whichincludes subtypes H3, H4, H7, H10, H14, and H15. In one embodiment, anantibody or binding fragment according to the invention is capable ofbinding to and/or neutralizing one or more influenza A virus group 1subtypes selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 andH17 and variants thereof and one or more influenza A virus group 2subtype selected from H3, H4, H7, H10, H14 and H15 and variants thereof.In another embodiment, an antibody or binding fragment according to theinvention is capable of binding to and/or neutralizing influenza A virusgroup 1 subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 andinfluenza A virus group 2 subtypes H3, H4, H7, H10, H14 and H15. Inanother embodiment, the antibody or binding fragment is capable ofbinding to and/or neutralizing group 1 subtypes H1, H2, H5, H6, and H9and group 2 subtypes H3 and H7. In a further embodiment, the antibody orbinding fragment is capable of binding to and/or neutralizing group 1subtypes H1, H2, H5 and H6 and group 2 subtypes H3 and H7.

The invention is based on isolation of a naturally-occurring humanmonoclonal antibody (mAb) from IgG memory B cells that were collectedfrom individual donors as starting materials. Optimization was used togenerate antibody variants with improved characteristics, as describedherein. The optimized antibody variants are not naturally occurring;they are generated using recombinant techniques. Antibody or fragmentsthereof of the invention bind to the stalk region of HA and neutralizeinfection of more than one subtype of influenza A virus, selected fromgroup 1 and group 2 subtypes, respectively. Antibodies of the invention,which are anti-Influenza A HA stalk-binding antibodies, demonstrated abroader breath of coverage or better neutralizing activity againstinfluenza A viruses compared to an antibody from the publishedliterature (Antibody FI6v4, described in WO2013/011347A1) and shown inTable 6 of Example 5. Additionally, antibodies of the invention may bemore effective than other mAb(s) in blocking HA maturation as shown inFIG. 1 of Example 6.

In some embodiments, the antibody or binding fragment thereof includes aset of six CDRs in which the set of six CDRs is selected from the groupconsisting of:

(a) HCDR1 of SEQ ID NO.: 3, HCDR2 of SEQ ID NO.: 4, HCDR3 of SEQ ID NO.:5, LCDR1 of SEQ ID NO.: 8, LCDR2 of SEQ ID NO.: 9 and LCDR3 of SEQ IDNO.: 10;

(b) HCDR1 of SEQ ID NO.: 13, HCDR2 of SEQ ID NO.: 14, HCDR3 of SEQ IDNO.: 15, LCDR1 of SEQ ID NO.: 18, LCDR2 of SEQ ID NO.: 19, LCDR3 of SEQID NO.: 20;

(c) HCDR1 of SEQ ID NO.: 23, HCDR2 of SEQ ID NO.: 24, HCDR3 of SEQ IDNO.: 25, LCDR1 of SEQ ID NO.: 28, LCDR2 of SEQ ID NO.: 29 and LCDR3 ofSEQ ID NO.: 30;

(d) HCDR1 of SEQ ID NO.: 33, HCDR2 of SEQ ID NO.: 34, HCDR3 of SEQ IDNO.: 35, LCDR1 of SEQ ID NO.: 38, LCDR2 of SEQ ID NO.: 39 and LCDR3 ofSEQ ID NO.: 40;

(e) HCDR1 of SEQ ID NO.: 43, HCDR2 of SEQ ID NO.: 44, HCDR3 of SEQ IDNO.: 45, LCDR1 of SEQ ID NO.: 48, LCDR2 of SEQ ID NO.: 49 and LCDR3 ofSEQ ID NO.: 50;

(f) HCDR1 of SEQ ID NO.: 53, HCDR2 of SEQ ID NO.: 54, HCDR3 of SEQ IDNO.: 55, LCDR1 of SEQ ID NO.: 58, LCDR2 of SEQ ID NO.: 59 and LCDR3 ofSEQ ID NO.: 60;

(g) HCDR1 of SEQ ID NO.: 63, HCDR2 of SEQ ID NO.: 64, HCDR3 of SEQ IDNO.: 65, LCDR1 of SEQ ID NO.: 68, LCDR2 of SEQ ID NO.: 69 and LCDR3 ofSEQ ID NO.: 70;

(h) HCDR1 of SEQ ID NO.: 73, HCDR2 of SEQ ID NO.: 74, HCDR3 of SEQ IDNO.: 75, LCDR1 of SEQ ID NO.: 78, LCDR2 of SEQ ID NO.: 79 and LCDR3 ofSEQ ID NO.: 80;

(i) HCDR1 of SEQ ID NO.: 83, HCDR2 of SEQ ID NO.: 84, HCDR3 of SEQ IDNO.: 85, LCDR1 of SEQ ID NO.: 88, LCDR2 of SEQ ID NO.: 89, LCDR3 of SEQID NO.: 90;

(j) HCDR1 of SEQ ID NO.: 93, HCDR2 of SEQ ID NO.: 94, HCDR3 of SEQ IDNO.: 95, LCDR1 of SEQ ID NO.: 98, LCDR2 of SEQ ID NO.: 99 and LCDR3 ofSEQ ID NO.: 100;

(k) HCDR1 of SEQ ID NO.: 103, HCDR2 of SEQ ID NO.: 104, HCDR3 of SEQ IDNO.: 105, LCDR1 of SEQ ID NO.: 108, LCDR2 of SEQ ID NO.: 109 and LCDR3of SEQ ID NO.: 110;

(l) HCDR1 of SEQ ID NO.: 113, HCDR2 of SEQ ID NO.: 114, HCDR3 of SEQ IDNO.: 115, LCDR1 of SEQ ID NO.: 118, LCDR2 of SEQ ID NO.: 119 and LCDR3of SEQ ID NO.: 110;

(m) HCDR1 of SEQ ID NO.: 123, HCDR2 of SEQ ID NO.: 124, HCDR3 of SEQ IDNO.: 125, LCDR1 of SEQ ID NO.: 128, LCDR2 of SEQ ID NO.: 129 and LCDR3of SEQ ID NO.: 130;

(n) HCDR1 of SEQ ID NO.: 133, HCDR2 of SEQ ID NO.: 134, HCDR3 of SEQ IDNO.: 135, LCDR1 of SEQ ID NO.: 138, LCDR2 of SEQ ID NO.: 139 and LCDR3of SEQ ID NO.: 140; and

(o) HCDR1 of SEQ ID NO.: 143, HCDR2 of SEQ ID NO.: 144, HCDR3 of SEQ IDNO.: 145, LCDR1 of SEQ ID NO.: 148, LCDR2 of SEQ ID NO.: 149 and LCDR3of SEQ ID NO.: 150;

(p) a set of six CDRS according to any one of (a) to (o) comprising oneor more amino acid substitutions, deletions or insertions;

(q) a set of six CDRS according to any one of (a) to (p) comprising 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, or 24 or 25 amino acid substitutions;

(r) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 accordingto any one of (a) to (q) comprising:

-   -   (i) a HCDR1 having an amino acid sequence identical to or        comprising 3 or fewer amino acid residue substitutions relative        to SEQ ID NO: 3;    -   (ii) a HCDR2 having an amino acid sequence identical to or        comprising 5 or fewer amino acid residue substitutions relative        to SEQ ID NO:4;    -   (iii) a HCDR3 having an amino acid sequence identical to or        comprising 6 or fewer amino acid residue substitutions relative        to SEQ ID NO:5;    -   (iv) a LCDR1 having an amino acid sequence identical to or        comprising 5 or fewer amino acid residue substitutions and/or        one deletion relative to SEQ ID NO:6;    -   (v) a LCDR2 having an amino acid sequence identical to or        comprising 5 or fewer amino acid residue substitutions relative        to SEQ ID NO:7; and    -   (vi) a LCDR3 having an amino acid sequence identical to or        comprising 1 or fewer amino acid residue substitutions relative        to SEQ ID NO:8;        (s) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3        according to any one of (a) to (r) comprising:    -   (i) a HCDR1 in which:    -   Kabat residue 31 is S,    -   Kabat residue 32 is N or Y,    -   Kabat residue 33 is N, S, or R,    -   Kabat residue 34 is A,    -   Kabat residue 35 is V or T,    -   Kabat residue 35A is W    -   Kabat residue 35B is N;    -   (ii) a HCDR2 in which:    -   Kabat residue 50 is R,    -   Kabat residue 51 is T,    -   Kabat residue 52 is Y,    -   Kabat residue 52A is Y,    -   Kabat residue 53 is R,    -   Kabat residue 54 is S,    -   Kabat residue 55 is K or G,    -   Kabat residue 56 is W,    -   Kabat residue 57 is Y,    -   Kabat residue 58 is N or Y,    -   Kabat residue 59 is D,    -   Kabat residue 60 is Y,    -   Kabat residue 61 is A,    -   Kabat residue 62 is E, V or d,    -   Kabat residue 63 is S or F,    -   Kabat residue 64 is V or L,    -   Kabat residue 65 is K;    -   (iii) a HCDR3 in which:    -   Kabat residue 95 is S or G,    -   Kabat residue 96 is G,    -   Kabat residue 97 is H,    -   Kabat residue 98 is I,    -   Kabat residue 99 is T,    -   Kabat residue 100 is V or E,    -   Kabat residue 100A is F,    -   Kabat residue 100B is G,    -   Kabat residue 100C is V or L,    -   Kabat residue 100D is N,    -   Kabat residue 100E is V or I,    -   Kabat residue 100F is D,    -   Kabat residue 100G is A,    -   Kabat residue 100F is F or Y,    -   Kabat residue 101 is D,    -   Kabat residue 102 is M, I or V;    -   (iv) a LCDR1 in which:    -   Kabat residue 24 is R,    -   Kabat residue 25 is T, A or absent,    -   Kabat residue 26 is S or A,    -   Kabat residue 27 is Q,    -   Kabat residue 28 is S or R,    -   Kabat residue 29 is L,    -   Kabat residue 30 is S, N or R    -   Kabat residue 31 is S,    -   Kabat residue 32 is Y,    -   Kabat residue 33 is L, T or D,    -   Kabat residue 34 is H;    -   (v) a LCDR2 in which:    -   Kabat residue 50 is A,    -   Kabat residue 51 is A, T or S,    -   Kabat residue 52 is S or T,    -   Kabat residue 53 is S or T,    -   Kabat residue 54 is L or R,    -   Kabat residue 55 is Q, L or G,    -   Kabat residue 56 is S; and,    -   (vi) a LCDR3 in which:    -   Kabat residue 89 is Q,    -   Kabat residue 90 is Q or L,    -   Kabat residue 91 is S,    -   Kabat residue 92 is R, and    -   Kabat residue 93 is T.

The invention provides antibodies and binding fragments thereofcomprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3,wherein the set of six CDRs is shown in Tables 11 and 13.

Variant antibody sequences of the invention may share 75% or more (e.g.,80%, 85%, 90%, 95%, 97%, 98%, 99% or more) amino acid sequence identitywith the sequences recited in the application. In some embodiments thesequence identity is calculated with regard to the full length of thereference sequence (i.e. the sequence recited in the application). Insome further embodiments, percentage identity, as referred to herein, isas determined using BLAST version 2.1.3 using the default parametersspecified by the NCBI (the National Center for BiotechnologyInformation; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap openpenalty=I 1 and gap extension penalty=I].

Variant antibodies are also included within the scope of the invention.Thus, variants of the sequences recited in the application are alsoincluded within the scope of the invention. Variants of the antibodysequences having improved affinity and/or potency may be obtained usingmethods known in the art and are included within the scope of theinvention. For example, amino acid substitutions may be used to obtainantibodies with further improved affinity. Alternatively, codonoptimization of the nucleotide sequence may be used to improve theefficiency of translation in expression systems for the production ofthe antibody. Further, polynucleotides comprising a sequence optimizedfor antibody specificity or neutralizing activity by the application ofa directed evolution method to any of the nucleic acid sequences of theinvention are also within the scope of the invention.

The invention provides an antibody or binding fragment thereof accordingto the invention comprising a VH having at least 75% identity and/or aVL having at least 75% identity to a VH and/or VL selected from thegroup consisting of:

(a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

(b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

(c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

(d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

(e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

(f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

(g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

(h) VH of SEQ ID NO.: 72 and VL of SEQ ID NO.: 77,

(i) VH of SEQ ID NO.: 82 and VL of SEQ ID NO.: 87,

(j) VH of SEQ ID NO.: 92 and VL of SEQ ID NO.: 97,

(k) VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107,

(l) VH of SEQ ID NO.: 112 and VL of SEQ ID NO.: 117,

(m) VH of SEQ ID NO.: 122 and VL of SEQ ID NO.: 127,

(n) VH of SEQ ID NO.: 132 and VL of SEQ ID NO.: 137,

(o) VH of SEQ ID NO.: 144 and VL of SEQ ID NO.: 147 and

(p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.

An antibody or binding fragment thereof according to the invention maycomprise a VH and a VL selected from the group consisting of:

(a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

(b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

(c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

(d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

(e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

(f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

(g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

(h) VH of SEQ ID NO.: 72 and VL of SEQ ID NO.: 77,

(i) VH of SEQ ID NO.: 82 and VL of SEQ ID NO.: 87,

(j) VH of SEQ ID NO.: 92 and VL of SEQ ID NO.: 97,

(k) VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107,

(l) VH of SEQ ID NO.: 112 and VL of SEQ ID NO.: 117,

(m) VH of SEQ ID NO.: 122 and VL of SEQ ID NO.: 127,

(n) VH of SEQ ID NO.: 132 and VL of SEQ ID NO.: 137,

(o) VH of SEQ ID NO.: 144 and VL of SEQ ID NO.: 147 and

(p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.

An antibody or binding fragment thereof according to the invention maybe selected from the group consisting of: an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahumanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linkedFv, a scFv, a single domain antibody, a diabody, a multispecificantibody, a dual-specific antibody, and a bispecific antibody.

An antibody or binding fragment thereof according to the invention maycomprise a VH comprising a human germline framework, preferably VH6-1and/or a VL comprising a human germline framework, preferably VK1-39.Preferably an antibody or binding fragment thereof according to theinvention comprises a VH comprising human germline framework VH6-1 and aVL comprising a human germline framework VK1-39. The VH6 framework israrely used in antibodies.

An antibody or binding fragment thereof according to the invention maycomprise an Fc region, preferably the antibody is an IgG1, IgG2 or IgG4or a binding fragment thereof.

In one embodiment, an antibody of the invention comprises a human IgGconstant domain having one or more amino acid substitutions relative toa wild-type human IgG constant domain. An antibody of the invention maycomprise a human IgG constant domain having the M252Y, S254T, and T256E(“YTE”) amino acid substitutions, wherein amino acid residues arenumbered according to the EU index as in Kabat.

The invention also provides an antibody to influenza A virus or abinding fragment thereof that is capable of binding to influenza A virushemagglutinin and neutralizing at least one group 1 subtype and at leastone group 2 subtype of influenza A virus characterized in that theantibody or binding fragment thereof competes for binding to influenza Avirus hemagglutinin with an antibody of the invention, described above.Accordingly, the invention comprises an antibody, or fragment thereof,that binds to the same epitope as an antibody of the invention, or anantibody that competes for binding with an antibody of the invention.

The invention further provides an isolated nucleic acid encoding anantibody or fragment thereof according to the invention. Preferably, thenucleic acid is a cDNA. The invention also includes nucleic acidsequences encoding part or all of the light and heavy chains and CDRs ofthe antibodies of the present invention. Thus, provided herein arenucleic acid sequences encoding part or all of the light and heavychains and CDRs of exemplary antibodies of the invention. The SEQ IDnumbers for the nucleic acid sequences encoding the CDRs, heavy chainand light chain variable regions of the exemplary antibodies of theinvention are provided. Due to the redundancy of the genetic code,variants of these sequences will exist that encode the same amino acidsequences.

The invention yet further provides a vector comprising an isolatednucleic acid according to the invention; preferably the vector is anexpression vector.

Additionally, the invention provides a host cell comprising an isolatednucleic acid or a vector according to the invention. Suitable host cellsinclude mammalian cell lines, such as those derived from HEK or CHOcells.

Further, the invention provides a method for manufacturing an antibodyor fragment of the invention comprising culturing a host cell of theinvention under conditions suitable for expression of the antibody orfragment thereof.

Such methods may further comprise isolating the antibody or fragmentthereof from the host cell culture and optionally formulating theisolated antibody or fragment into a composition.

The invention yet further provides a composition comprising an antibodyor fragment thereof according to the invention and a pharmaceuticallyacceptable carrier.

Also provided by the invention is a composition comprising an antibodyor fragment thereof according to the invention, histidine and NaCl at apH in the range of from about 5.5 to about 6.5, preferably at about pH6.0; yet more preferably comprising an antibody or fragment thereofaccording to the invention, about 20 to about 30 mM histidine and about0.1 to about 0.2 M NaCl, at a pH in the range of from about 5.5 to about6.5, preferably at about pH 6.0; most preferably comprising 25 mM Hisand 0.15M NaCl at a pH in the range of from about 5.5 to about 6.5, forexample, at about pH 6.0

Additionally, the invention provides:

-   -   an antibody or fragment thereof according to the invention for        use in the prophylaxis or treatment of influenza A infection in        a subject;    -   the use of an antibody or fragment thereof according to the        invention in the manufacture of a medicament for the prophylaxis        or treatment of Influenza A infection in a subject;    -   a method for prophylaxis or treatment of Influenza A infection        in a subject comprising administration of an antibody or        fragment thereof according to the invention;    -   the use of an antibody or fragment thereof according to the        invention to prevent the pH-triggered fusion step for Influenza        A viral entry into cells; or    -   the use of an antibody or fragment thereof according to the        invention to inhibit Influenza A virus HA maturation.

Exemplary antibodies of the invention include, but are not limited to:Antibody 3, Antibody 5, Antibody 6, Antibody 8, Antibody 10, Antibody11, Antibody 12, Antibody 13, Antibody 14, and Antibody 15.

The invention also provides the use of an antibody or binding fragmentthereof according to the invention in in vitro diagnosis of influenza Ainfection in a subject.

DETAILED DESCRIPTION Introduction

The present invention provides antibodies, including human forms, aswell as fragments, derivatives/conjugates and compositions thereof thatbind to Influenza A virus hemagglutinin (HA) stalk and neutralizeinfluenza A virus infection group 1 and group 2 subtypes as describedherein; such anti-influenza A virus HA stalk antibodies are referred toherein as antibodies of the invention.

As used herein, the term “neutralize” refers to the ability of anantibody, or binding fragment thereof, to bind to an infectious agent,such as influenza A virus, and reduce the biological activity, forexample, virulence, of the infectious agent. The minimal requirement forneutralization is the ability for the antibody, or binding fragmentthereof, to bind to the infectious agent. In one embodiment, theantibody or binding fragment thereof of the invention immunospecificallybinds at least one specified epitope or antigenic determinant of theInfluenza A virus. In a more particular embodiment, the antibody orbinding fragment thereof of the invention immunospecifically binds atleast one specified epitope or antigenic determinant of the Influenza Avirus HA stalk protein.

An antibody can neutralize the activity of an infectious agent, such asInfluenza A virus at various points during the lifecycle of the virus.For example, an antibody may interfere with viral attachment to a targetcell by interfering with the interaction of the virus and one or morecell surface receptors. Alternately, an antibody may interfere with oneor more post-attachment interactions of the virus with its receptors,for example, by interfering with viral internalization byreceptor-mediated endocytosis.

In one embodiment, the antibody or binding fragment thereof neutralizesthe activity of Influenza A by interfering with the fusion process, forexample, by interfering with fusion of the viral and endosomalmembranes. In another embodiment, the antibody or binding fragmentthereof interferes with protease mediated cleavage of HA0, thusinterfering with viral maturation and the formation of the HA2 viralfusion peptide. For example, in one embodiment, the antibody or bindingfragment thereof interferes with protease mediated HA0 cleavage,necessary for activation of the Influenza A virus.

As used herein, the terms “antibody” and “antibodies”, also known asimmunoglobulins, encompass monoclonal antibodies (including full-lengthmonoclonal antibodies), human antibodies, humanized antibodies, camelidantibodies, chimeric antibodies, single-chain Fvs (scFv), single-chainantibodies, single domain antibodies, domain antibodies, Fab fragments,F(ab′)2 fragments, antibody fragments that exhibit the desiredbiological activity (e.g. the antigen binding portion), disulfide-linkedFvs (dsFv), and anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id antibodies to antibodies of the invention), intrabodies, andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain at least oneantigen-binding site. Immunoglobulin molecules can be of any isotype(e.g., IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or allotype (e.g., Gm, e.g., G1 m(f, z, a orx), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2 or 3)).

Human antibodies are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Each heavy chain has at one end a variable domain(VH) followed by a number of constant domains (CH). Each light chain hasa variable domain at one end (VL) and a constant domain (CL) at itsother end; the constant domain of the light chain is aligned with thefirst constant domain of the heavy chain, and the light chain variabledomain is aligned with the variable domain of the heavy chain. Lightchains are classified as either lambda chains or kappa chains based onthe amino acid sequence of the light chain constant region. The variabledomain of a kappa light chain may also be denoted herein as VK.

The antibodies of the invention include full length or intact antibody,antibody fragments, including antigen binding fragments, native sequenceantibody or amino acid variants, human, humanized, post-translationallymodified, chimeric or fusion antibodies, immunoconjugates, andfunctional fragments thereof. The antibodies can be modified in the Fcregion to provide desired effector functions or serum half-life. Asdiscussed in more detail in the sections below, with the appropriate Fcregions, the naked antibody bound on the cell surface can inducecytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC)or by recruiting complement in complement dependent cytotoxicity (CDC),or by recruiting nonspecific cytotoxic cells that express one or moreeffector ligands that recognize bound antibody on the Influenza A virusHA stalk and subsequently cause phagocytosis of the cell in antibodydependent cell-mediated phagocytosis (ADCP), or some other mechanism.Alternatively, where it is desirable to eliminate or reduce effectorfunction, so as to minimize side effects or therapeutic complications,certain other Fc regions may be used. The Fc region of the antibodies ofthe invention can be modified to increase the binding affinity for FcRnand thus increase serum half-life. Alternatively, the Fc region can beconjugated to PEG or albumin to increase the serum half-life, or someother conjugation that results in the desired effect.

The present anti-Influenza A virus HA stalk antibodies are useful fordiagnosing, preventing, treating and/or alleviating one or more symptomsof the Influenza A virus infection in a mammal.

The invention provides a composition comprising an anti-Influenza Avirus HA stalk antibody of the invention and a carrier. For the purposesof preventing or treating Influenza A virus infection, compositions canbe administered to the patient in need of such treatment. The inventionalso provides formulations comprising an anti-Influenza A virus HA stalkantibody of the invention and a carrier. In one embodiment, theformulation is a therapeutic formulation comprising a pharmaceuticallyacceptable carrier.

In certain embodiments the invention provides methods useful forpreventing or treating Influenza A infection in a mammal, comprisingadministering a therapeutically effective amount of the antibody to themammal. The antibody therapeutic compositions can be administered shortterm (acutely), chronically, or intermittently as directed by physician.

In certain embodiments the invention also provides articles ofmanufacture comprising at least an anti-Influenza A virus HA stalkantibody, such as sterile dosage forms and kits. Kits can be providedwhich contain the antibodies for detection and quantitation of InfluenzaA virus in vitro, e.g. in an ELISA or a Western blot. Such antibodyuseful for detection may be provided with a label such as a fluorescentor radiolabel.

TERMINOLOGY

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisinvention.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The numbering of amino acids in the variable domain, complementaritydetermining region (CDRs) and framework regions (FR), of an antibodyfollow, unless otherwise indicated, the Kabat definition as set forth inKabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insertion (residue 52a according to Kabat) after residue 52 of H2and inserted residues (e.g. residues 82a, 82b, and 82c, etc., accordingto Kabat) after heavy chain FR residue 82. The Kabat numbering ofresidues may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence. Maximal alignment of framework residues frequentlyrequires the insertion of “spacer” residues in the numbering system, tobe used for the Fv region. In addition, the identity of certainindividual residues at any given Kabat site number may vary fromantibody chain to antibody chain due to interspecies or allelicdivergence.

Anti-Influenza a Virus HA Stalk Antibodies

In certain embodiments, the antibodies are isolated and/or purifiedand/or pyrogen free antibodies. The term “purified” as used herein,refers to other molecules, e.g., polypeptide, nucleic acid molecule thathave been identified and separated and/or recovered from a component ofits natural environment. Thus, in one embodiment the antibodies of theinvention are purified antibodies wherein they have been separated fromone or more components of their natural environment. The term “isolatedantibody” as used herein refers to an antibody which is substantiallyfree of other antibody molecules having different antigenicspecificities (e.g., an isolated antibody that specifically binds toInfluenza A virus HA stalk is substantially free of antibodies thatspecifically bind antigens other than those of Influenza A virus HAstalk). Thus, in one embodiment the antibodies of the invention areisolated antibodies wherein they have been separated from antibodieswith a different specificity. Typically an isolated antibody is amonoclonal antibody. Moreover, an isolated antibody of the invention maybe substantially free of one or more other cellular materials and/orchemicals and is herein referred to an isolated and purified antibody.In one embodiment of the invention, a combination of “isolated”monoclonal antibodies relates to antibodies having differentspecificities and being combined in a well-defined composition. Methodsof production and purification/isolation of antibodies are describedbelow in more detail.

The isolated antibodies of the present invention comprise antibody aminoacid sequences disclosed herein encoded by any suitable polynucleotide,or any isolated or formulated antibody.

The antibodies of the invention immunospecifically bind at least onespecified epitope specific to the Influenza A virus HA stalk protein.The term “epitope” as used herein refers to a protein determinantcapable of binding to an antibody. Epitopes usually include chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

In one embodiment, the antibody or binding fragment thereof binds to anepitope that is conserved among at least H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15, H16 or H17 or all influenza A HAsubtypes. In another embodiment, the antibody or binding fragmentthereof binds to an epitope that is conserved among one or more, or atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 influenza A virus group 1subtypes selected from H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 andone or more, or at least 1, 2, 3, 4, 5, or 6 group 2 subtypes selectedfrom H3, H4, H7, H10, H14 and H15.

In one embodiment, the antibody or binding fragment thereof binds atleast 17H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, H16 or H17 or all influenza A subtypes with an EC₅₀ of betweenabout 0.01 ug/ml and about 5 ug/ml, or between about 0.01 ug/ml andabout 0.5 ug/ml, or between about 0.01 ug/ml and about 0.1 ug/ml, orless than about 5 ug/ml, 1 ug/ml, 0.5 ug/ml, 0.1 ug/ml, or 0.05 ug/ml.In another embodiment, the antibody or binding fragment thereof bindsone or more, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 influenza Avirus group 1 subtypes selected from H1, H2, H5, H6, H8, H9, H11, H12,H13 and H16 and one or more, or at least 1, 2, 3, 4, 5, or 6 group 2subtypes selected from H3, H4, H7, H10, H14 and H15 with an EC₅₀ ofbetween about 0.01 ug/ml and about 5 ug/ml, or between about 0.01 ug/mland about 0.5 ug/ml, or between about 0.01 ug/ml and about 0.1 ug/ml, orless than about 5 ug/ml, 1 ug/ml, 0.5 ug/ml, 0.1 ug/ml, or 0.05 ug/ml.

In one embodiment, the antibody or binding fragment thereof recognizesan epitope that is either a linear epitope, or continuous epitope. Inanother embodiment, the antibody or binding fragment thereof recognizesa non-linear or conformational epitope. In one embodiment, the epitopeis located in the highly conserved stalk region of HA2. In a moreparticular embodiment, the antibody or binding fragment binds to aconformational epitope in the highly conserved stalk region of HA2. Inone embodiment, the epitope includes one or more amino acids selectedfrom: positions 18, 19, 42, 45 in the stalk region of HA2 (positions arenumbered according to H3 numbering system as described in Weiss et al.,J. Mol. Biol. (1990) 212, 737-761 (1990)) as contact residues. In a moreparticular embodiment, the epitope includes one or more amino acidsselected from 18, 19, 42 and 45 in the stalk region of HA2 as contactresidues. In a further embodiment, the epitope includes amino acids 18,19, 42 and 45 in the stalk region of HA2 as contact residues. In yet afurther embodiment, the epitope includes amino acids 18, 19, and 42 inthe stalk region of HA2 as contact residues.

The epitope or epitopes recognized by the antibody or binding fragmentthereof of the invention may have a number of uses. For example, theepitope in purified or synthetic form can be used to raise immuneresponses (i.e., as a vaccine, or for the production of antibodies forother uses) or for screening sera for antibodies that immunoreact withthe epitope. In one embodiment, an epitope recognized by the antibody orbinding fragment thereof of the invention, or an antigen having such anepitope may be used as a vaccine for raising an immune response. Inanother embodiment, the antibodies and binding fragments of theinvention can be used to monitor the quality of vaccines, for example,by determining whether the antigen in a vaccine contains the correctimmunogenic epitope in the correct conformation.

Variable Regions

As used herein, the term “parent antibody” refers to an antibody whichis encoded by an amino acid sequence used for the preparation of thevariant or derivative, defined herein. The parent polypeptide maycomprise a native antibody sequence (i.e., a naturally occurring,including a naturally occurring allelic variant) or an antibody sequencewith pre-existing amino acid sequence modifications (such as otherinsertions, deletions and/or substitutions) of a naturally occurringsequence. The parent antibody may be a humanized antibody or a humanantibody. In specific embodiments, antibodies of the invention arevariants of the parent antibody. As used herein, the term “variant”refers to an antibody, which differs in amino acid sequence from a“parent” antibody amino acid sequence by virtue of addition, deletionand/or substitution of one or more amino acid residue(s) in the parentantibody sequence.

The antigen-binding portion of an antibody comprises one or morefragments of an antibody that retain the ability to specifically bind toan antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. Antigen-bindingportions can be produced by recombinant DNA techniques, or by enzymaticor chemical cleavage of intact immunoglobulins.

Antibodies of the invention comprise at least one antigen bindingdomain, comprising a VH and a VL domain described herein.

In certain embodiments, the purified antibodies comprise a VH and/or VLthat has a given percent identify to at least one of the VH and/or VLsequences disclosed in Table 1 As used herein, the term “percent (%)sequence identity”, also including “homology” is defined as thepercentage of amino acid residues or nucleotides in a candidate sequencethat are identical with the amino acid residues or nucleotides in thereference sequences, such as parent antibody sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Optimal alignment of thesequences for comparison may be produced, besides manually, by means ofthe local homology algorithm of Smith and Waterman, 1981, Ads App. Math.2, 482, by means of the local homology algorithm of Neddleman andWunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity searchmethod of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444,or by means of computer programs which use these algorithms (GAP,BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.).

Antibodies of the invention may comprise a VH amino acid sequence havingat least 65%, 70%, 75%, 80%, 85%, 90%, 95% or having 100% identity tothe VH amino acid sequences described herein. The antibodies may have aVH amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or having 100% identity to the amino acid sequence ofthe VH amino acid sequences described herein.

Antibodies of the invention may comprise a VL amino acid sequence havingat least 65%, 70%, 75%, 80%, 85%, 90%, 95% or having 100% identity tothe VL amino acid sequences described herein. The antibodies may have aVL amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or having 100% identity to the VL amino acidsequences described herein.

Antibodies within the scope of the of the invention are capable ofneutralizing one or more group 1 subtype and one or more group 2 subtypeof Influenza A virus, as described herein.

Complementarity Determining Regions (CDRs)

While the variable domain (VH and VL) comprises the antigen-bindingregion; the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in segments calledComplementarity Determining Regions (CDRs), both in the light chain (VLor VK) and the heavy chain (VH) variable domains. The more highlyconserved portions of the variable domains are called the frameworkregions (FR). The variable domains of native heavy and light chains eachcomprise four FR, largely adopting a β-sheet configuration, connected bythree CDRs, which form loops connecting, and in some cases forming partof, the β-sheet structure. The CDRs in each chain are held together inclose proximity by the FR and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies(see, Kabat et al., Supra). The three CDRs of the heavy chain aredesignated CDR-H1, CDR-H2, and CDR-H3, and the three CDRs of the lightchain are designated CDR-L1, CDR-L2, and CDR-L3. The Kabat numberingsystem is used herein. As such, CDR-H1 begins at approximately aminoacid 31 (i.e., approximately 9 residues after the first cysteineresidue), includes approximately 5-7 amino acids, and ends at the nexttyrosine residue. CDR-H2 begins at the fifteenth residue after the endof CDR-H1, includes approximately 16-19 amino acids, and ends at thenext arginine or lysine residue. CDR-H3 begins at approximately thethirty third amino acid residue after the end of CDR-H2; includes 3-25amino acids; and ends at the sequence W-G-X-G, where X is any aminoacid. CDR-L1 begins at approximately residue 24 (i.e., following acysteine residue); includes approximately 10-17 residues; and ends atthe next tyrosine residue. CDR-L2 begins at approximately the sixteenthresidue after the end of CDR-L1 and includes approximately 7 residues.CDR-L3 begins at approximately the thirty third residue after the end ofCDR-L2; includes approximately 7-11 residues and ends at the sequenceF-G-X-G, where X is any amino acid. Note that CDRs vary considerablyfrom antibody to antibody (and by definition will not exhibit homologywith the Kabat consensus sequences).

The present invention encompasses neutralizing anti-Influenza A HA stalkantibodies comprising amino acids in a sequence that is substantiallythe same as an amino acid sequence described herein. Amino acidsequences that are substantially the same as the sequences describedherein include sequences comprising conservative amino acidsubstitutions, as well as amino acid deletions and/or insertions in anamino acid sequence of for example, Antibody 11, Antibody 12, Antibody13, Antibody 14 or Antibody 15, or in an amino acid sequence shown inSEQ ID NOs: 102, 112, 122, 132, or 142. A conservative amino acidsubstitution refers to the replacement of a first amino acid by a secondamino acid that has chemical and/or physical properties (e.g, charge,structure, polarity, hydrophobicity/hydrophilicity) that are similar tothose of the first amino acid. Conservative substitutions includereplacement of one amino acid by another within the following groups:lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate(E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine(Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine(I), proline (P), phenylalanine (F), tryptophan (W), methionine (M),cysteine (C) and glycine (G); F, W and Y; C, S and T.

Framework Regions

The variable domains of the heavy and light chains each comprise fourframework regions (FR1, FR2, FR3, FR4), which are the more highlyconserved portions of the variable domains. The four FRs of the heavychain are designated FR-H1, FR-H2, FR-H3 and FR-H4, and the four FRs ofthe light chain are designated FR-L1, FR-L2, FR-L3 and FR-L4. The Kabatnumbering system is used herein, See Table 1, Kabat et al, Supra. Assuch, FR-H1 begins at position 1 and ends at approximately amino acid30, FR-H2 is approximately from amino acid 36 to 49, FR-H3 isapproximately from amino acid 66 to 94 and FR-H4 is approximately aminoacid 103 to 113. FR-L1 begins at amino acid 1 and ends at approximatelyamino acid 23, FR-L2 is approximately from amino acid 35 to 49, FR-L3 isapproximately from amino acid 57 to 88 and FR-L4 is approximately fromamino acid 98 to 107. In certain embodiments the framework regions maycontain substitutions according to the Kabat numbering system, e.g.,insertion at 106A in FR-L1. In addition to naturally occurringsubstitutions, one or more alterations (e.g., substitutions) of FRresidues may also be introduced in an antibody of the invention,provided it retains neutralizing ability. In certain embodiments, theseresult in an improvement or optimization in the binding affinity of theantibody for Influenza A virus HA stalk. Examples of framework regionresidues to modify include those which non-covalently bind antigendirectly (Amit et al., Science, 233:747-753 (1986)); interactwith/effect the conformation of a CDR (Chothia et al., J. Mol. Biol.,196:901-917 (1987)); and/or participate in the VL-VH interface (U.S.Pat. No. 5,225,539).

In another embodiment the FR may comprise one or more amino acid changesfor the purposes of “germlining”. For example, the amino acid sequencesof selected antibody heavy and light chains are compared to germlineheavy and light chain amino acid sequences and where certain frameworkresidues of the selected VL and/or VH chains differ from the germlineconfiguration (e.g., as a result of somatic mutation of theimmunoglobulin genes used to prepare the phage library), it may bedesirable to “back-mutate” the altered framework residues of theselected antibodies to the germline configuration (i.e., change theframework amino acid sequences of the selected antibodies so that theyare the same as the germline framework amino acid sequences). Such“back-mutation” (or “germlining”) of framework residues can beaccomplished by standard molecular biology methods for introducingspecific mutations (e.g., site-directed mutagenesis; PCR-mediatedmutagenesis, and the like).

Nucleotide Sequences Encoding Antibodies of the Invention

In addition to the amino acid sequences described above, the inventionfurther provides nucleotide sequences corresponding to the amino acidsequences and encoding for the human antibodies of the invention. In oneembodiment, the invention provides polynucleotides comprising anucleotide sequence encoding an antibody described herein or fragmentsthereof. These include, but are not limited to, nucleotide sequencesthat code for the above referenced amino acid sequences. Thus, thepresent invention also provides polynucleotide sequences encoding VH andVL framework regions including CDRs and FRs of antibodies describedherein as well as expression vectors for their efficient expression incells (e.g. mammalian cells). Methods of making the antibodies usingpolynucleotides are described below in more detail.

The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedherein, to polynucleotides that encode an antibody of the inventiondescribed herein. The term “stringency” as used herein refers toexperimental conditions (e.g. temperature and salt concentration) of ahybridization experiment to denote the degree of homology between theprobe and the filter bound nucleic acid; the higher the stringency, thehigher percent homology between the probe and filter bound nucleic acid.

Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., highly stringent conditions such as hybridization tofilter-bound DNA in 6×SSC at about 45° C. followed by one or more washesin 0.1×SSC/0.2% SDS at about 65° C., or any other stringenthybridization conditions known to those skilled in the art (see, forexample, Ausubel, F. M. et al., eds. 1989 Current Protocols in MolecularBiology, vol. 1, Green Publishing Associates, Inc. and John Wiley andSons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

Substantially identical sequences may be polymorphic sequences, i.e.,alternative sequences or alleles in a population. An allelic differencemay be as small as one base pair. Substantially identical sequences mayalso comprise mutagenized sequences, including sequences comprisingsilent mutations. A mutation may comprise one or more residue changes, adeletion of one or more residues, or an insertion of one or moreadditional residues.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

A polynucleotide encoding an antibody may also be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the immunoglobulinmay be chemically synthesized or obtained from a suitable source (e.g.,an antibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

Binding Characteristics

As described above, the anti-Influenza A virus HA stalk antibodies ofthe invention immunospecifically bind at least one specified epitope orantigenic determinants of the Influenza A virus HA stalk protein,peptide, subunit, fragment, portion or any combination thereof eitherexclusively or preferentially with respect to other polypeptides. Theterm “epitope” or “antigenic determinant” as used herein refers to aprotein determinant capable of binding to an antibody, wherein the term“binding” herein preferably relates to a specific binding. These proteindeterminants or epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have a specific three dimensional structural characteristics, aswell as specific charge characteristics. Conformational andnon-conformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. The term “discontinuous epitope” as used herein, refers to aconformational epitope on a protein antigen which is formed from atleast two separate regions in the primary sequence of the protein.

The interactions between antigens and antibodies are the same as forother non-covalent protein-protein interactions. In general, four typesof binding interactions exist between antigens and antibodies: (i)hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forcesbetween Lewis acids and Lewis bases, and (iv) hydrophobic interactions.Hydrophobic interactions are a major driving force for theantibody-antigen interaction, and are based on repulsion of water bynon-polar groups rather than attraction of molecules (Tanford, 1978).However, certain physical forces also contribute to antigen-antibodybinding, for example, the fit or complimentary of epitope shapes withdifferent antibody binding sites. Moreover, other materials and antigensmay cross-react with an antibody, thereby competing for available freeantibody.

Measurement of the affinity constant and specificity of binding betweenantigen and antibody is a pivotal element in determining the efficacy ofprophylactic, therapeutic, diagnostic and research methods using theantibodies of the invention. “Binding affinity” generally refers to thestrength of the sum total of the noncovalent interactions between asingle binding site of a molecule (e.g., an antibody) and its bindingpartner (e.g., an antigen). Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a1:1 interaction between members of a binding pair (e.g., antibody andantigen). The affinity of a molecule X for its partner Y can generallybe represented by the equilibrium dissociation constant (Kd), which iscalculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol Biol 293:865-881. Affinity can be measured by commonmethods known in the art, including those described and exemplifiedherein. An example of a commercially available system for kineticcharacterization includes the OCTET® family of instruments. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention.

Determination of binding affinity can be measured using the specifictechniques described further in the Example section, and methods wellknown in the art. One such method includes measuring the disassociationconstant “Kd” by a radiolabeled antigen binding assay (RIA) performedwith the Fab version of an antibody of interest and its antigen asdescribed by the following assay that measures solution binding affinityof Fabs for antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (H 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbant plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of an anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., 65 hours) to insure that equilibriumis reached. Thereafter, the mixtures are transferred to the captureplate for incubation at room temperature (e.g., for one hour). Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays.

In another instance the Kd value may be measured by using surfaceplasmon resonance assays using a BIAcore™-2000 or a BIAcore™-3000(BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5chips at ^(˜)10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 110 mM sodium acetate, pH 4.8, into 5 ug/ml(^(˜)0.2 uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, IM ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneously fitting the association and dissociationsensorgram.

If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surface plasmon resonanceassay above, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stir red cuvette. An “on-rate” or “rate ofassociation” or “association rate” or “k_(on)” according to thisinvention can also be determined with the same surface plasmon resonancetechnique described above using a BIAcore™-2000 or a BIAcore™-3000(BIAcore, Inc., Piscataway, N.J.) as described above.

Methods and reagents suitable for determination of bindingcharacteristics of an antibody of the present invention, or analtered/mutant derivative thereof (discussed below), are known in theart and/or are commercially available (U.S. Pat. Nos. 6,849,425;6,632,926; 6,294,391; 6,143,574). Moreover, equipment and softwaredesigned for such kinetic analyses are commercially available (e.g.Biacore® A100, and Biacore® 2000 instruments; Biacore International AB,Uppsala, Sweden).

In one embodiment, antibodies of the present invention, includingbinding fragments or variants thereof, may also be described orspecified in terms of their binding affinity for Influenza A viruspolypeptides. Typically, antibodies with high affinity have Kd of lessthan 10⁻⁷ M. In one embodiment, antibodies or binding fragments thereofbind Influenza A polypeptides, or fragments or variants thereof, with adissociation constant or Kd of less than or equal to 5×10⁻⁷ M, 10⁻⁷ M,5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ Mor 10⁻¹⁵ M. Influenza A polypeptides can include HA polypeptides. In amore particular embodiment, antibodies or binding fragments thereof bindInfluenza A polypeptides, or fragments or variants thereof, with adissociation constant or Kd of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M or 10⁻¹² M. The invention encompassesantibodies that bind Influenza A polypeptides with a dissociationconstant or Kd that is within a range between any of the individualrecited values.

In another embodiment, antibodies or binding fragments thereof of theinvention bind Influenza A polypeptides or fragments or variants thereofwith an off rate (k_(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻²sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹, 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷sec⁻¹. In a more particular embodiment, antibodies or binding fragmentsthereof of the invention bind Influenza A polypeptides or fragments orvariants thereof with an off rate (k_(off)) less than or equal to 5×10⁻⁴sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹. The invention also encompassesantibodies that bind Influenza A polypeptides with an off rate (k_(off))that is within a range between any of the individual recited values.

In another embodiment, antibodies or binding fragments thereof of theinvention bind Influenza A polypeptides or fragments or variants thereofwith an on rate (k_(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹,5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹, 5×10⁴ M⁻¹ sec⁻¹, 10⁵ M⁻¹ sec⁻¹, 5×10⁵M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec-1, 5×10⁶ M⁻¹ sec⁻¹, 10⁷ M⁻¹ sec-1, or 5×10⁷ M⁻¹sec⁻¹. In a more particular embodiment, antibodies or binding fragmentsthereof of the invention bind Influenza A polypeptides or fragments orvariants thereof with an on rate (k_(on)) greater than or equal to 10⁵M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec-1, 5×10⁶ M⁻¹ sec⁻¹, 10⁷ M⁻¹sec⁻¹ or 5×10⁷ M⁻¹ sec⁻¹. The invention encompasses antibodies that bindInfluenza A polypeptides with on rate (k_(on)) that is within a rangebetween any of the individual recited values.

In one embodiment, a binding assay may be performed either as directbinding assays or as competition-binding assays. Binding can be detectedusing standard ELISA or standard Flow Cytometry assays. In a directbinding assay, a candidate antibody is tested for binding to its cognateantigen. Competition-binding assay, on the other hand, assess theability of a candidate antibody to compete with a known antibody orother compound that binds to the Influenza A virus HA stalk. In generalany method that permits the binding of an antibody with the Influenza Avirus HA stalk that can be detected is encompassed with the scope of thepresent invention for detecting and measuring the bindingcharacteristics of the antibodies. One of skill in the art willrecognize these well-known methods and for this reason are not providedin detail here. These methods are also utilized to screen a panel ofantibodies for those providing the desired characteristics.

An antibody of the invention immunospecifically binds to Influenza Avirus HA stalk and is capable of neutralizing Influenza A virusinfection. Neutralization assays can be performed as described herein inthe Examples section or using other methods known in the art. The term“inhibitory concentration 50%” (abbreviated as “IC₅₀”) represents theconcentration of an inhibitor (e.g., an antibody of the invention) thatis required for 50% neutralization of Influenza A virus. It will beunderstood by one of ordinary skill in the art that a lower IC₅₀ valuecorresponds to a more potent inhibitor.

In one embodiment, an antibody or binding fragment thereof according tothe invention has a neutralizing potency expressed as 50% inhibitoryconcentration (IC₅₀ ug/ml) in the range of from about 0.01 ug/ml toabout 50 ug/ml, or in the range of from about 0.01 ug/ml to about 5ug/ml of antibody, or in the range of from about 0.01 ug/ml to about 0.1ug/ml of antibody for neutralization of influenza A virus in amicroneutralization assay. The highest concentration of antibody used inmicroneutralization assay described herein was 50 ug/ml. The highpotency of antibodies of the invention means that lower concentrationsof antibody can be used to attain 50% neutralization of influenza Avirus.

In certain embodiments, the antibodies of the invention may induce celldeath. An antibody which “induces cell death” is one which causes aviable cell to become nonviable. Cell death in vitro may be determinedin the absence of complement and immune effector cells to distinguishcell death induced by antibody-dependent cell-mediated cytotoxicity(ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay forcell death may be performed using heat inactivated serum (i.e., in theabsence of complement) and in the absence of immune effector cells. Todetermine whether the antibody is able to induce cell death, loss ofmembrane integrity as evaluated by uptake of propidium iodide (PI),trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)), 7AAD orother methods well known in the art can be assessed relative tountreated cells.

In a specific embodiment, the antibodies of the invention may inducecell death via apoptosis. An antibody which “induces apoptosis” is onewhich induces programmed cell death as determined by binding of annexinV, fragmentation of DNA, cell shrinkage, dilation of endoplasmicreticulum, cell fragmentation, and/or formation of membrane vesicles(called apoptotic bodies). Various methods are available for evaluatingthe cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNAfragmentation can be evaluated through DNA laddering; andnuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the antibodywhich induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay.

In another specific embodiment, the antibodies of the invention mayinduce cell death via antibody-dependent cellular cytotoxicity (ADCC)and/or complement-dependent cell-mediated cytotoxicity (CDC) and/orantibody dependent cell-mediated phagocytosis (ADCP). Expression of ADCCactivity and CDC activity of the human IgG1 subclass antibodiesgenerally involves binding of the Fc region of the antibody to areceptor for an antibody (hereinafter referred to as “FcγR”) existing onthe surface of effector cells such as killer cells, natural killer cellsor activated macrophages. Various complement components can be bound.Regarding the binding, it has been suggested that several amino acidresidues in the hinge region and the second domain of C region(hereinafter referred to as “Cγ2 domain”) of the antibody are important(Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995), ChemicalImmunology, 65, 88 (1997)) and that a sugar chain in the Cγ2 domain(Chemical Immunology, 65, 88 (1997)) is also important.

To assess ADCC activity of an antibody of interest, an in vitro ADCCassay can be used, such as that described in U.S. Pat. No. 5,500,362.The assay may also be performed using a commercially available kit, e.g.CytoTox 96 ® (Promega). Useful effector cells for such assays include,but are not limited to peripheral blood mononuclear cells (PBMC),Natural Killer (NK) cells, and NK cell lines. NK cell lines expressing atransgenic Fc receptor (e.g. CD16) and associated signaling polypeptide(e.g. FC_(ε)RI-γ) may also serve as effector cells (WO 2006/023148). Forexample, the ability of any particular antibody to mediate lysis bycomplement activation and/or ADCC can be assayed. The cells of interestare grown and labeled in vitro; the antibody is added to the cellculture in combination with immune cells which may be activated by theantigen antibody complexes; i.e., effector cells involved in the ADCCresponse. The antibody can also be tested for complement activation. Ineither case, cytolysis is detected by the release of label from thelysed cells. The extent of cell lysis may also be determined bydetecting the release of cytoplasmic proteins (e.g. LDH) into thesupernatant. In fact, antibodies can be screened using the patient's ownserum as a source of complement and/or immune cells. Antibodies that arecapable of mediating human ADCC in the in vitro test can then be usedtherapeutically in that particular patient. ADCC activity of themolecule of interest may also be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci.(USA) 95:652-656 (1998). Moreover, techniques for modulating (i.e.,increasing or decreasing) the level of ADCC, and optionally CDCactivity, of an antibody are well-known in the art (e.g., U.S. Pat. Nos.5,624,821; 6,194,551; 7,317,091). Antibodies of the present inventionmay be capable or may have been modified to have the ability of inducingADCC and/or CDC. Assays to determine ADCC function can be practicedusing human effector cells to assess human ADCC function. Such assaysmay also include those intended to screen for antibodies that induce,mediate, enhance, block cell death by necrotic and/or apoptoticmechanisms. Such methods including assays utilizing viable dyes, methodsof detecting and analyzing caspases, and assays measuring DNA breaks canbe used to assess the apoptotic activity of cells cultured in vitro withan antibody of interest.

Production of Antibodies

The following describes exemplary techniques for the production of theantibodies useful in the present invention.

Monoclonal Antibodies

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma (Kohler et al., Nature,256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981), recombinant, and phage display technologies, or a combinationthereof. The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneous orisolated antibodies, e.g., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to polyclonal antibody preparations whichinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against the samedeterminant on the antigen. In addition to their specificity, monoclonalantibodies are advantageous in that they may be synthesizeduncontaminated by other antibodies. The modifier “monoclonal” is not tobe construed as requiring production of the antibody by any particularmethod. Following is a description of representative methods forproducing monoclonal antibodies which is not intended to be limiting andmay be used to produce, for example, monoclonal mammalian, chimeric,humanized, human, domain, diabodies, vaccibodies, linear andmultispecific antibodies.

Hybridoma Techniques

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In thehybridoma method, mice or other appropriate host animals, such ashamster, are immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the antigen used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent orfusion partner, such as polyethylene glycol, to form a hybridoma cell(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). In certain embodiments, the selected myelomacells are those that fuse efficiently, support stable high-levelproduction of antibody by the selected antibody-producing cells, and aresensitive to a selective medium that selects against the unfusedparental cells. In one aspect, the myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA. Humanmyeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Supra). Suitable culture media for this purpose include, for example,D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grownin vivo as ascites tumors in an animal e.g., by i.p. injection of thecells into mice.

The monoclonal antibodies secreted by the sub-clones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, affinity tags, hydroxylapatitechromatography, gel electrophoresis, dialysis, etc. Exemplarypurification methods are described in more detail below.

Recombinant DNA Techniques

Methods for producing and screening for specific antibodies usingrecombinant DNA technology are routine and well known in the art (e.g.U.S. Pat. No. 4,816,567). DNA encoding the monoclonal antibodies may bereadily isolated and/or sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of murine antibodies). Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese Hamster Ovary (CHO) cells, or myeloma cells that do nototherwise produce antibody protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) andPluckthun, Immunol. Revs., 130:151-188 (1992). As described below forantibodies generated by phage display and humanization of antibodies,DNA or genetic material for recombinant antibodies can be obtained fromsource(s) other than hybridomas to generate antibodies of the invention.

Recombinant expression of an antibody or variant thereof generallyrequires construction of an expression vector containing apolynucleotide that encodes the antibody. The invention, thus, providesreplicable vectors comprising a nucleotide sequence encoding an antibodymolecule, a heavy or light chain of an antibody, a heavy or light chainvariable domain of an antibody or a portion thereof, or a heavy or lightchain CDR, operably linked to a promoter. Such vectors may include thenucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., U.S. Pat. Nos. 5,981,216; 5,591,639; 5,658,759 and5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy, the entire lightchain, or both the entire heavy and light chains.

Once the expression vector is transferred to a host cell by conventionaltechniques, the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, the invention includes hostcells containing a polynucleotide encoding an antibody of the inventionor fragments thereof, or a heavy or light chain thereof, or portionthereof, or a single-chain antibody of the invention, operably linked toa heterologous promoter. In certain embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

Mammalian cell lines available as hosts for expression of recombinantantibodies are well known in the art and include many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney293 cells, and a number of other cell lines. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the antibody or portion thereofexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0(a murine myeloma cell line that does not endogenously produce anyfunctional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells.Human cell lines developed by immortalizing human lymphocytes can beused to recombinantly produce monoclonal antibodies. The human cell linePER.C6. (Crucell, Netherlands) can be used to recombinantly producemonoclonal antibodies.

Additional cell lines which may be used as hosts for expression ofrecombinant antibodies include, but are not limited to, insect cells(e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S.cerevisiae, Pichia, U.S. Pat. No. 7,326,681; etc), plants cells(US20080066200); and chicken cells (WO2008142124).

In certain embodiments, antibodies of the invention are expressed in acell line with stable expression of the antibody. Stable expression canbe used for long-term, high-yield production of recombinant proteins.For example, cell lines which stably express the antibody molecule maybe generated. Host cells can be transformed with an appropriatelyengineered vector comprising expression control elements (e.g.,promoter, enhancer, transcription terminators, polyadenylation sites,etc.), and a selectable marker gene. Following the introduction of theforeign DNA, cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells that stably integrated the plasmid into their chromosomesto grow and form foci which in turn can be cloned and expanded into celllines. Methods for producing stable cell lines with a high yield arewell known in the art and reagents are generally available commercially.

In certain embodiments, antibodies of the invention are expressed in acell line with transient expression of the antibody. Transienttransfection is a process in which the nucleic acid introduced into acell does not integrate into the genome or chromosomal DNA of that cell.It is in fact maintained as an extra-chromosomal element, e.g. as anepisome, in the cell. Transcription processes of the nucleic acid of theepisome are not affected and a protein encoded by the nucleic acid ofthe episome is produced.

The cell line, either stable or transiently transfected, is maintainedin cell culture medium and conditions well known in the art resulting inthe expression and production of monoclonal antibodies. In certainembodiments, the mammalian cell culture media is based on commerciallyavailable media formulations, including, for example, DMEM or Ham's F12.In other embodiments, the cell culture media is modified to supportincreases in both cell growth and biologic protein expression. As usedherein, the terms “cell culture medium,” “culture medium,” and “mediumformulation” refer to a nutritive solution for the maintenance, growth,propagation, or expansion of cells in an artificial in vitro environmentoutside of a multicellular organism or tissue. Cell culture medium maybe optimized for a specific cell culture use, including, for example,cell culture growth medium which is formulated to promote cellulargrowth, or cell culture production medium which is formulated to promoterecombinant protein production. The terms nutrient, ingredient, andcomponent are used interchangeably herein to refer to the constituentsthat make up a cell culture medium.

In one embodiment, the cell lines are maintained using a fed batchmethod. As used herein, “fed batch method,” refers to a method by whicha fed batch cell culture is supplied with additional nutrients afterfirst being incubated with a basal medium. For example, a fed batchmethod may comprise adding supplemental media according to a determinedfeeding schedule within a given time period. Thus, a “fed batch cellculture” refers to a cell culture wherein the cells, typicallymammalian, and culture medium are supplied to the culturing vesselinitially and additional culture nutrients are fed, continuously or indiscrete increments, to the culture during culturing, with or withoutperiodic cell and/or product harvest before termination of culture.

The cell culture medium used and the nutrients contained therein areknown to one of skill in the art. In one embodiment, the cell culturemedium comprises a basal medium and at least one hydrolysate, e.g.,soy-based hydrolysate, a yeast-based hydrolysate, or a combination ofthe two types of hydrolysates resulting in a modified basal medium. Inanother embodiment, the additional nutrients may include only a basalmedium, such as a concentrated basal medium, or may include onlyhydrolysates, or concentrated hydrolysates. Suitable basal mediainclude, but are not limited to Dulbecco's Modified Eagle's Medium(DMEM), DME/F12, Minimal Essential Medium (MEM), Basal Medium Eagle(BME), RPMI 1640, F-10, F-12, α-Minimal Essential Medium (α-MEM),Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g., CHOprotein free medium (Sigma) or EX-CELL™ 325 PF CHO Serum-Free Medium forCHO Cells Protein-Free (SAFC Bioscience), and Iscove's ModifiedDulbecco's Medium. Other examples of basal media which may be used inthe invention include BME Basal Medium (Gibco-Invitrogen; see alsoEagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's ModifiedEagle Medium (DMEM, powder) (Gibco-Invitrogen (#31600); see alsoDulbecco and Freeman (1959) Virology 8, 396; Smith et al. (1960)Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93);CMRL 1066 Medium (Gibco-Invitrogen (#11530); see also Parker R. C. et al(1957) Special Publications, N.Y. Academy of Sciences, 5, 303).

The basal medium may be serum-free, meaning that the medium contains noserum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or anyother animal-derived serum known to one skilled in the art) or animalprotein free media or chemically defined media.

The basal medium may be modified in order to remove certainnon-nutritional components found in standard basal medium, such asvarious inorganic and organic buffers, surfactant(s), and sodiumchloride. Removing such components from basal cell medium allows anincreased concentration of the remaining nutritional components, and mayimprove overall cell growth and protein expression. In addition, omittedcomponents may be added back into the cell culture medium containing themodified basal cell medium according to the requirements of the cellculture conditions. In certain embodiments, the cell culture mediumcontains a modified basal cell medium, and at least one of the followingnutrients, an iron source, a recombinant growth factor; a buffer; asurfactant; an osmolarity regulator; an energy source; and non-animalhydrolysates. In addition, the modified basal cell medium may optionallycontain amino acids, vitamins, or a combination of both amino acids andvitamins. In another embodiment, the modified basal medium furthercontains glutamine, e.g, L-glutamine, and/or methotrexate.

Antibody production can be conducted in large quantity by a bioreactorprocess using fed-batch, batch, perfusion or continuous feed bioreactormethods known in the art. Large-scale bioreactors have at least 1000liters of capacity, preferably about 1,000 to 100,000 liters ofcapacity. These bioreactors may use agitator impellers to distributeoxygen and nutrients. Small scale bioreactors refers generally to cellculturing in no more than approximately 100 liters in volumetriccapacity, and can range from about 1 liter to about 100 liters.Alternatively, single-use bioreactors (SUB) may be used for eitherlarge-scale or small-scale culturing.

Temperature, pH, agitation, aeration and inoculum density will varydepending upon the host cells used and the recombinant protein to beexpressed. For example, a recombinant protein cell culture may bemaintained at a temperature between 30 and 45° C. The pH of the culturemedium may be monitored during the culture process such that the pHstays at an optimum level, which may be for certain host cells, within apH range of 6.0 to 8.0. An impellor driven mixing may be used for suchculture methods for agitation. The rotational speed of the impellor maybe approximately 50 to 200 cm/sec tip speed, but other airlift or othermixing/aeration systems known in the art may be used, depending on thetype of host cell being cultured. Sufficient aeration is provided tomaintain a dissolved oxygen concentration of approximately 20% to 80%air saturation in the culture, again, depending upon the selected hostcell being cultured. Alternatively, a bioreactor may sparge air oroxygen directly into the culture medium. Other methods of oxygen supplyexist, including bubble-free aeration systems employing hollow fibermembrane aerators.

Phage Display Techniques

Monoclonal antibodies or antibody fragments can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).In such methods antibodies can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fromhuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612), examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display libraries can be found in, for example, U.S.Pat. Nos. 6,248,516; 6,545,142; 6,291,158; 6,291,159; 6,291,160;6,291,161; 6,680,192; 5,969,108; 6,172,197; 6,806,079; 5,885,793;6,521,404; 6,544,731; 6,555,313; 6,593,081; 6,582,915; 7,195,866. Thus,these techniques are viable alternatives to traditional monoclonalantibody hybridoma techniques for generation and isolation of monoclonalantibodies.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen-binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, humanizedantibodies, or any other desired antigen binding fragment, and expressedin any desired host, including mammalian cells, insect cells, plantcells, yeast, and bacteria, e.g., as described in detail below. Forexample, techniques to recombinantly produce Fab, Fab′ and F(ab′)2fragments can also be employed using methods known in the art such asthose disclosed in PCT publication WO 92/22324; Mullinax et al.,BioTechniques 12(6):864-869 (1992); and Better et al., Science240:1041-1043 (1988).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498. Thus, techniques described above and those well known in theart can be used to generate recombinant antibodies wherein the bindingdomain, e.g. ScFv, was isolated from a phage display library.

Antibody Purification and Isolation

Once an antibody molecule has been produced by recombinant or hybridomaexpression, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigens Protein A or Protein G, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies of the present invention or fragments thereof may be fused toheterologous polypeptide sequences (referred to herein as “tags”) tofacilitate purification.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology, 10:163-167 (1992) describe a procedure forisolating antibodies which are secreted into the periplasmic space of E.coli. Where the antibody is secreted into the medium, supernatants fromsuch expression systems are generally first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. A protease inhibitorsuch as PMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, ion exchange chromatography, gel electrophoresis,dialysis, and/or affinity chromatography either alone or in combinationwith other purification steps. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody and will be understood by oneof skill in the art. The matrix to which the affinity ligand is attachedis most often agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH₃ domain, the Bakerbond ABX resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin, SEPHAROSE chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, and performed at low salt concentrations (e.g.,from about 0-0.25 M salt).

Thus, in certain embodiments is provided antibodies of the inventionthat are substantially purified/isolated. In one embodiment, theseisolated/purified recombinantly expressed antibodies may be administeredto a patient to mediate a prophylactic or therapeutic effect. Aprophylactic is a medication or a treatment designed and used to preventa disease, disorder or infection from occurring. A therapeutic isconcerned specifically with the treatment of a particular disease,disorder or infection. A therapeutic dose is the amount needed to treata particular disease, disorder or infection. In another embodiment theseisolated/purified antibodies may be used to diagnose Influenza A virusinfection.

Human Antibodies

Human antibodies can be generated using methods well known in the art.Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient.

Human antibodies can be derived by in vitro methods. Suitable examplesinclude but are not limited to phage display (MedImmune (formerly CAT),Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerlyProliferon), Affimed) ribosome display (MedImmune (formerly CAT)), yeastdisplay, and the like. The phage display technology (See e.g., U.S. Pat.No. 5,969,108) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Immunoglobulin genes undergo various modifications during maturation ofthe immune response, including recombination between V, D and J genesegments, isotype switching, and hypermutation in the variable regions.Recombination and somatic hypermutation are the foundation forgeneration of antibody diversity and affinity maturation, but they canalso generate sequence liabilities that may make commercial productionof such immunoglobulins as therapeutic agents difficult or increase theimmunogenicity risk of the antibody. In general, mutations in CDRregions are likely to contribute to improved affinity and function,while mutations in framework regions may increase the risk ofimmunogenicity. This risk can be reduced by reverting frameworkmutations to germline while ensuring that activity of the antibody isnot adversely impacted. The diversification processes may also generatesome structural liabilities or these structural liabilities may existwithin germline sequences contributing to the heavy and light chainvariable domains. Regardless of the source, it may be desirable toremove potential structural liabilities that may result in instability,aggregation, heterogeneity of product, or increased immunogenicity.Examples of undesirable liabilities include unpaired cysteines (whichmay lead to disulfide bond scrambling, or variable sulfhydryl adductformation), N-linked glycosylation sites (resulting in heterogeneity ofstructure and activity), as well as deamidation (e.g. NG, NS),isomerization (DG), oxidation (exposed methionine), and hydrolysis (DP)sites.

Accordingly, in order to reduce the risk of immunogenicity and improvepharmaceutical properties, it may be desirable to revert a frameworksequence to germline, revert a CDR to germline, and/or remove astructural liability.

Thus, in one embodiment, where a particular antibody differs from itsrespective germline sequence at the amino acid level, the antibodysequence can be mutated back to the germline sequence. Such correctivemutations can occur at one, two, three or more positions, or acombination of any of the mutated positions, using standard molecularbiological techniques.

Antibody Fragments

In certain embodiments, the present antibodies are antibody fragments orantibodies comprising these fragments. The antibody fragment comprises aportion of the full length antibody, which generally is the antigenbinding or variable region thereof. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)₂, Fd and Fv fragments. Diabodies; linearantibodies (U.S. Pat. No. 5,641,870) and single-chain antibodymolecules.

Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies using techniques well known in the art. However, thesefragments can now be produced directly by recombinant host cells. Fab,Fv and scFv antibody fragments can all be expressed in and secreted fromE. coli, thus allowing the facile production of large amounts of thesefragments. In one embodiment, the antibody fragments can be isolatedfrom the antibody phage libraries discussed above. Alternatively,Fab′-SH fragments can also be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology, 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single-chain Fv fragment (scFv). In certainembodiments, the antibody is not a Fab fragment. Fv and scFv are theonly species with intact combining sites that are devoid of constantregions; thus, they are suitable for reduced nonspecific binding duringin vivo use. scFv fusion proteins may be constructed to yield fusion ofan effector protein at either the amino or the carboxy terminus of anscFv.

In certain embodiments, the present antibodies are domain antibodies,e.g., antibodies containing the small functional binding units ofantibodies, corresponding to the variable regions of the heavy (VH) orlight (VL) chains of human antibodies. Examples of domain antibodiesinclude, but are not limited to, those of Domantis (see, for example,WO04/058821; WO04/081026; WO04/003019; WO03/002609; U.S. Pat. Nos.6,291,158; 6,582,915; 6,696,245; and 6,593,081).

In certain embodiments of the invention, the present antibodies arelinear antibodies. Linear antibodies comprise a pair of tandem Fdsegments (VH-CH1-VH-CH1) which form a pair of antigen-binding regions.See, Zapata et al., Protein Eng., 8(10):1057-1062 (1995).

Other Amino Acid Sequence Modifications

In addition to the above described human, humanized and/or chimericantibodies, the present invention also encompasses further modificationsand, their variants and fragments thereof, of the antibodies of theinvention comprising one or more amino acid residues and/or polypeptidesubstitutions, additions and/or deletions in the variable light (VL)domain and/or variable heavy (VH) domain and/or Fc region and posttranslational modifications. Included in these modifications areantibody conjugates wherein an antibody has been covalently attached toa moiety. Moieties suitable for attachment to the antibodies include butare not limited to, proteins, peptides, drugs, labels, and cytotoxins.These changes to the antibodies may be made to alter or fine tune thecharacteristics (biochemical, binding and/or functional) of theantibodies as is appropriate for treatment and/or diagnosis of InfluenzaA infection. Methods for forming conjugates, making amino acid and/orpolypeptide changes and post-translational modifications are well knownin the art, some of which are detailed below.

Amino acid changes to the antibodies necessarily results in sequencesthat are less than 100% identical to the above identified antibodysequences or parent antibody sequence. In certain embodiments, in thiscontext, the antibodies many have about 25% to about 95% sequenceidentity to the amino acid sequence of either the heavy or light chainvariable domain of an antibody as described herein. Thus, in oneembodiment a modified antibody may have an amino acid sequence having atleast 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% amino acid sequence identity or similarity with the amino acidsequence of either the heavy or light chain variable domain of anantibody as described herein. In another embodiment, an altered antibodymay have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequenceidentity or similarity with the amino acid sequence of the heavy orlight chain CDR1, CDR2, or CDR3 of an antibody as described herein. Inanother embodiment, an altered antibody may have an amino acid sequencehaving at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% amino acid sequence identity or similarity with theamino acid sequence of the heavy or light chain FR1, FR2, FR3 or FR4 ofan antibody as described herein.

In certain embodiments, altered antibodies are generated by one or moreamino acid alterations (e.g., substitutions, deletion and/or additions)introduced in one or more of the variable regions of the antibody. Inanother embodiment, the amino acid alterations are introduced in theframework regions. One or more alterations of framework region residuesmay result in an improvement in the binding affinity of the antibody forthe antigen. This may be especially true when these changes are made tohumanized antibodies wherein the framework region may be from adifferent species than the CDR regions. Examples of framework regionresidues to modify include those which non-covalently bind antigendirectly (Amit et al., Science, 233:747-753 (1986)); interactwith/effect the conformation of a CDR (Chothia et al., J. Mol. Biol.,196:901-917 (1987)); and/or participate in the VL-VH interface (U.S.Pat. Nos. 5,225,539 and 6,548,640). In one embodiment, from about one toabout five framework residues may be altered. Sometimes, this may besufficient to yield an antibody mutant suitable for use in preclinicaltrials, even where none of the hypervariable region residues have beenaltered. Normally, however, an altered antibody will comprise additionalhypervariable region alteration(s).

One useful procedure for generating altered antibodies is called“alanine scanning mutagenesis” (Cunningham and Wells, Science,244:1081-1085 (1989)). In this method, one or more of the hypervariableregion residue(s) are replaced by alanine or polyalanine residue(s) toalter the interaction of the amino acids with the target antigen. Thosehypervariable region residue(s) demonstrating functional sensitivity tothe substitutions then are refined by introducing additional or othermutations at or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. The Ala-mutantsproduced this way are screened for their biological activity asdescribed herein.

In certain embodiments the substitutional variant involves substitutingone or more hypervariable region residues of a parent antibody (e.g. ahumanized or human antibody). Generally, the resulting variant(s)selected for further development will have improved biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display (Hawkins et al., J.Mol. Biol., 254:889-896 (1992) and Lowman et al., Biochemistry,30(45):10832-10837 (1991)). Briefly, several hypervariable region sites(e.g., 6-7 sites) are mutated to generate all possible amino acidsubstitutions at each site. The antibody mutants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage-displayed mutants are then screened for their biologicalactivity (e.g., binding affinity) as herein disclosed.

Mutations in antibody sequences may include substitutions, deletions,including internal deletions, additions, including additions yieldingfusion proteins, or conservative substitutions of amino acid residueswithin and/or adjacent to the amino acid sequence, but that result in a“silent” change, in that the change produces a functionally-equivalentantibody. Conservative amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. In addition, glycine and proline are residues that caninfluence chain orientation. Non-conservative substitutions will entailexchanging a member of one of these classes for a member of anotherclass. Furthermore, if desired, non-classical amino acids or chemicalamino acid analogs can be introduced as a substitution or addition intothe antibody sequence. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, and amino acid analogs in general.

In another embodiment, any cysteine residue not involved in maintainingthe proper conformation of the antibody also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)may be added to the antibody to improve its stability (particularlywhere the antibody is an antibody fragment such as an Fv fragment).

Variant Fc Regions

It is known that variants of the Fc region (e.g., amino acidsubstitutions and/or additions and/or deletions) enhance or diminisheffector function of the antibody (See e.g., U.S. Pat. Nos. 5,624,821;5,885,573; 6,538,124; 7,317,091; 5,648,260; 6,538,124; WO 03/074679; WO04/029207; WO 04/099249; WO 99/58572; US Publication No. 2006/0134105;2004/0132101; 2006/0008883) and may alter the pharmacokinetic properties(e.g. half-life) of the antibody (see, U.S. Pat. Nos. 6,277,375 and7,083,784). Thus, in certain embodiments, the antibodies of theinvention comprise an altered Fc region (also referred to herein as“variant Fc region”) in which one or more alterations have been made inthe Fc region in order to change functional and/or pharmacokineticproperties of the antibodies. Such alterations may result in a decreaseor increase of Clq binding and complement dependent cytotoxicity (CDC)or of FcγR binding, for IgG, and antibody-dependent cellularcytotoxicity (ADCC), or antibody dependent cell-mediated phagocytosis(ADCP). The present invention encompasses the antibodies describedherein with variant Fc regions wherein changes have been made to finetune the effector function, enhancing or diminishing, providing adesired effector function. Accordingly, the antibodies of the inventioncomprise a variant Fc region (i.e., Fc regions that have been altered asdiscussed below). Antibodies of the invention comprising a variant Fcregion are also referred to here as “Fc variant antibodies.” As usedherein native refers to the unmodified parental sequence and theantibody comprising a native Fc region is herein referred to as a“native Fc antibody”. Fc variant antibodies can be generated by numerousmethods well known to one skilled in the art. Non-limiting examplesinclude, isolating antibody coding regions (e.g., from hybridoma) andmaking one or more desired substitutions in the Fc region of theisolated antibody coding region. Alternatively, the antigen-bindingportion (e.g., variable regions) of an antibody may be sub-cloned into avector encoding a variant Fc region. In one embodiment, the variant Fcregion exhibits a similar level of inducing effector function ascompared to the native Fc region. In another embodiment, the variant Fcregion exhibits a higher induction of effector function as compared tothe native Fc. Some specific embodiments of variant Fc regions aredetailed infra. Methods for measuring effector function are well knownin the art.

The effector function of an antibody is modified through changes in theFc region, including but not limited to, amino acid substitutions, aminoacid additions, amino acid deletions and changes in post-translationalmodifications to Fc amino acids (e.g. glycosylation). The methodsdescribed below may be used to fine tune the effector function of apresent antibody, a ratio of the binding properties of the Fc region forthe FcR (e.g., affinity and specificity), resulting in a therapeuticantibody with the desired properties.

It is understood that the Fc region as used herein includes thepolypeptides comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain. Thus Fc refers to the lasttwo constant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as set forth in Kabat. Fc may refer to thisregion in isolation, or this region in the context of an antibody,antibody fragment, or Fc fusion protein. Polymorphisms have beenobserved at a number of different Fc positions, including but notlimited to positions 270, 272, 312, 315, 356, and 358 as numbered by theEU index, and thus slight differences between the presented sequence andsequences in the prior art may exist.

In one embodiment, Fc variant antibodies exhibit altered bindingaffinity for one or more Fc receptors including, but not limited toFcRn, FcγRI (CD64) including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII(CD32 including isoforms FcγRIIA, FcγRIIB, and FcγRIIC); and FcγRIII(CD16, including isoforms FcγRIIIA and FcγRIIIB) as compared to annative Fc antibody.

In one embodiment, an Fc variant antibody has enhanced binding to one ormore Fc ligand relative to a native Fc antibody. In another embodiment,the Fc variant antibody exhibits increased or decreased affinity for anFc ligand that is at least 2 fold, or at least 3 fold, or at least 5fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or atleast 30 fold, or at least 40 fold, or at least 50 fold, or at least 60fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, orat least 100 fold, or at least 200 fold, or is between 2 fold and 10fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, orbetween 75 fold and 200 fold, or between 100 and 200 fold, more or lessthan a native Fc antibody. In another embodiment, Fc variant antibodiesexhibit affinities for an Fc ligand that are at least 90%, at least 80%,at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, atleast 20%, at least 10%, or at least 5% more or less than an native Fcantibody. In certain embodiments, an Fc variant antibody has increasedaffinity for an Fc ligand. In other embodiments, an Fc variant antibodyhas decreased affinity for an Fc ligand.

In a specific embodiment, an Fc variant antibody has enhanced binding tothe Fc receptor FcγRIIIA. In another specific embodiment, an Fc variantantibody has enhanced binding to the Fc receptor FcγRIIB. In a furtherspecific embodiment, an Fc variant antibody has enhanced binding to boththe Fc receptors FcγRIIIA and FcγRIIB. In certain embodiments, Fcvariant antibodies that have enhanced binding to FcγRIIIA do not have aconcomitant increase in binding the FcγRIIB receptor as compared to anative Fc antibody. In a specific embodiment, an Fc variant antibody hasreduced binding to the Fc receptor FcγRIIIA. In a further specificembodiment, an Fc variant antibody has reduced binding to the Fcreceptor FcγRIIB. In still another specific embodiment, an Fc variantantibody exhibiting altered affinity for FcγRIIIA and/or FcγRIIB hasenhanced binding to the Fc receptor FcRn. In yet another specificembodiment, an Fc variant antibody exhibiting altered affinity forFcγRIIIA and/or FcγRIIB has altered binding to C1q relative to a nativeFc antibody.

In one embodiment, Fc variant antibodies exhibit affinities for FcγRIIIAreceptor that are at least 2 fold, or at least 3 fold, or at least 5fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or atleast 30 fold, or at least 40 fold, or at least 50 fold, or at least 60fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, orat least 100 fold, or at least 200 fold, or are between 2 fold and 10fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, orbetween 75 fold and 200 fold, or between 100 and 200 fold, more or lessthan an native Fc antibody. In another embodiment, Fc variant antibodiesexhibit affinities for FcγRIIIA that are at least 90%, at least 80%, atleast 70%, at least 60%, at least 50%, at least 40%, at least 30%, atleast 20%, at least 10%, or at least 5% more or less than an native Fcantibody.

In one embodiment, Fc variant antibodies exhibit affinities for FcγRIIBreceptor that are at least 2 fold, or at least 3 fold, or at least 5fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or atleast 30 fold, or at least 40 fold, or at least 50 fold, or at least 60fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, orat least 100 fold, or at least 200 fold, or are between 2 fold and 10fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, orbetween 75 fold and 200 fold, or between 100 and 200 fold, more or lessthan an native Fc antibody. In another embodiment, Fc variant antibodiesexhibit affinities for FcγRIIB that are at least 90%, at least 80%, atleast 70%, at least 60%, at least 50%, at least 40%, at least 30%, atleast 20%, at least 10%, or at least 5% more or less than an native Fcantibody.

In one embodiment, Fc variant antibodies exhibit increased or decreasedaffinities to C1q relative to a native Fc antibody. In anotherembodiment, Fc variant antibodies exhibit affinities for C1q receptorthat are at least 2 fold, or at least 3 fold, or at least 5 fold, or atleast 7 fold, or a least 10 fold, or at least 20 fold, or at least 30fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, orat least 70 fold, or at least 80 fold, or at least 90 fold, or at least100 fold, or at least 200 fold, or are between 2 fold and 10 fold, orbetween 5 fold and 50 fold, or between 25 fold and 100 fold, or between75 fold and 200 fold, or between 100 and 200 fold, more or less than annative Fc antibody. In another embodiment, Fc variant antibodies exhibitaffinities for C1q that are at least 90%, at least 80%, at least 70%, atleast 60%, at least 50%, at least 40%, at least 30%, at least 20%, atleast 10%, or at least 5% more or less than an native Fc antibody. Instill another specific embodiment, an Fc variant antibody exhibitingaltered affinity for Ciq has enhanced binding to the Fc receptor FcRn.In yet another specific embodiment, an Fc variant antibody exhibitingaltered affinity for C1q has altered binding to FcγRIIIA and/or FcγRIIBrelative to a native Fc antibody.

It is well known in the art that antibodies are capable of directing theattack and destruction through multiple processes collectively known inthe art as antibody effector functions. One of these processes, known as“antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enables these cytotoxic effector cells tobind specifically to an antigen-bearing cells and subsequently kill thecells with cytotoxins. Specific high-affinity IgG antibodies directed tothe surface of cells “arm” the cytotoxic cells and are required for suchkilling. Lysis of the cell is extracellular, requires directcell-to-cell contact, and does not involve complement.

Another process encompassed by the term effector function is complementdependent cytotoxicity (hereinafter referred to as “CDC”) which refersto a biochemical event of cell destruction by the complement system. Thecomplement system is a complex system of proteins found in normal bloodplasma that combines with antibodies to destroy pathogenic bacteria andother foreign cells.

Still another process encompassed by the term effector function isantibody dependent cell-mediated phagocytosis (ADCP) which refers to acell-mediated reaction wherein nonspecific cytotoxic cells that expressone or more effector ligands recognize bound antibody on a cell andsubsequently cause phagocytosis of the cell.

It is contemplated that Fc variant antibodies are characterized by invitro functional assays for determining one or more FcγR mediatedeffector cell functions. In certain embodiments, Fc variant antibodieshave similar binding properties and effector cell functions in in vivomodels (such as those described and disclosed herein) as those in invitro based assays. However, the present invention does not exclude Fcvariant antibodies that do not exhibit the desired phenotype in in vitrobased assays but do exhibit the desired phenotype in vivo.

In certain embodiments, an antibody comprising an Fc variant hasenhanced cytotoxicity or phagocytosis activity (e.g., ADCC, CDC andADCP) relative to an antibody comprising a native Fc region. In aspecific embodiment, an Fc variant antibody has cytotoxicity orphagocytosis activity that is at least 2 fold, or at least 3 fold, or atleast 5 fold or at least 10 fold or at least 50 fold or at least 100fold, or at least 200 fold, or is between 2 fold and 10 fold, or between5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 foldand 200 fold, or between 100 and 200 fold, greater than that of a nativeFc antibody. Alternatively, an Fc variant antibody has reducedcytotoxicity or phagocytosis activity relative to a native Fc antibody.In a specific embodiment, an Fc variant antibody has cytotoxicity orphagocytosis activity that is at least 2 fold, or at least 3 fold, or atleast 5 fold or at least 10 fold or at least 50 fold or at least 100fold, or at least 200 fold, or is between 2 fold and 10 fold, or between5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 foldand 200 fold, or between 100 and 200 fold, lower than that of a nativeFc antibody.

In certain embodiments, Fc variant antibodies exhibit decreased ADCCactivities as compared to a native Fc antibody. In another embodiment,Fc variant antibodies exhibit ADCC activities that are at least 2 fold,or at least 3 fold, or at least 5 fold or at least 10 fold or at least50 fold or at least 100 fold, or at least 200 fold, or is between 2 foldand 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100fold, or between 75 fold and 200 fold, or between 100 and 200 fold, lessthan that of a native Fc antibody. In still another embodiment, Fcvariant antibodies exhibit ADCC activities that are reduced by at least10%, or at least 20%, or by at least 30%, or by at least 40%, or by atleast 50%, or by at least 60%, or by at least 70%, or by at least 80%,or by at least 90%, or by at least 100%, or by at least 200%, or by atleast 300%, or by at least 400%, or by at least 500%, relative to anative Fc antibody. In certain embodiments, Fc variant antibodies haveno detectable ADCC activity. In specific embodiments, the reductionand/or ablatement of ADCC activity may be attributed to the reducedaffinity Fc variant antibodies exhibit for Fc ligands and/or receptors.

In an alternative embodiment, Fc variant antibodies exhibit increasedADCC activities as compared to a native Fc antibody. In anotherembodiment, Fc variant antibodies exhibit ADCC activities that are atleast 2 fold, or at least 3 fold, or at least 5 fold or at least 10 foldor at least 50 fold or at least 100 fold greater than that of a nativeFc antibody. In still another embodiment, Fc variant antibodies exhibitADCC activities that are increased by at least 10%, or at least 20%, orby at least 30%, or by at least 40%, or by at least 50%, or by at least60%, or by at least 70%, or by at least 80%, or by at least 90%, or byat least 100%, or by at least 200%, or by at least 300%, or by at least400%, or by at least 500% relative to a native Fc antibody. In specificembodiments, the increased ADCC activity may be attributed to theincreased affinity Fc variant antibodies exhibit for Fc ligands and/orreceptors.

In a specific embodiment, an Fc variant antibody has enhanced binding tothe Fc receptor FcγRIIIA and has enhanced ADCC activity relative to anative Fc antibody. In other embodiments, the Fc variant antibody hasboth enhanced ADCC activity and enhanced serum half-life relative to anative Fc antibody. In another specific embodiment, an Fc variantantibody has reduced binding to the Fc receptor FcγRIIIA and has reducedADCC activity relative to a native Fc antibody. In other embodiments,the Fc variant antibody has both reduced ADCC activity and enhancedserum half-life relative to a native Fc antibody.

In certain embodiments, the cytotoxicity is mediated by CDC wherein theFc variant antibody has either enhanced or decreased CDC activityrelative to a native Fc antibody. The complement activation pathway isinitiated by the binding of the first component of the complement system(C1q) to a molecule, an antibody for example, complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., 1996, J. Immunol. Methods, 202:163, may beperformed.

In one embodiment, antibodies of the invention exhibit increased CDCactivity as compared to a native Fc antibody. In another embodiment, Fcvariant antibodies exhibit CDC activity that is at least 2 fold, or atleast 3 fold, or at least 5 fold or at least 10 fold or at least 50 foldor at least 100 fold, or at least 200 fold, or is between 2 fold and 10fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, orbetween 75 fold and 200 fold, or between 100 and 200 fold more than thatof an native Fc antibody. In still another embodiment, Fc variantantibodies exhibit CDC activity that is increased by at least 10%, or atleast 20%, or by at least 30%, or by at least 40%, or by at least 50%,or by at least 60%, or by at least 70%, or by at least 80%, or by atleast 90%, or by at least 100%, or by at least 200%, or by at least300%, or by at least 400%, or by at least 500% relative to a native Fcantibody. In specific embodiments, the increase of CDC activity may beattributed to the increased affinity Fc variant antibodies exhibit forC1q.

Antibodies of the invention may exhibit increased CDC activity ascompared to a native Fc antibody by virtue of COMPLEGENT® Technology(Kyowa Hakko Kirin Co., Ltd.), which enhances one of the majormechanisms of action of an antibody, CDC. With an approach calledisotype chimerism, in which portions of IgG3, an antibody's isotype, areintroduced into corresponding regions of IgG1, the standard isotype fortherapeutic antibodies, COMPLEGENT® Technology significantly enhancesCDC activity beyond that of either IgG1 or IgG3, while retaining thedesirable features of IgG1, such as ADCC, PK profile and Protein Abinding. In addition, it can be used together with POTELLIGENT®Technology, creating an even superior therapeutic Mab (ACCRETAMAB®) withenhanced ADCC and CDC activities

Fc variant antibody of the invention may have enhanced ADCC activity andenhanced serum half-life relative to a native Fc antibody.

Fc variant antibody of the invention may CDC activity and enhanced serumhalf life relative to a native Fc antibody.

Fc variant antibody of the invention may have enhanced ADCC activity,enhanced CDC activity and enhanced serum half-life relative to a nativeFc antibody.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. The term“antibody half-life” as used herein means a pharmacokinetic property ofan antibody that is a measure of the mean survival time of antibodymolecules following their administration. Antibody half-life can beexpressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body (or other mammal) ora specific compartment thereof, for example, as measured in serum, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

The increase in half-life allows for the reduction in amount of druggiven to a patient as well as reducing the frequency of administration.To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Alternatively, antibodies of the invention with increased half-lives maybe generated by modifying amino acid residues identified as involved inthe interaction between the Fc and the FcRn receptor (see, for examples,U.S. Pat. Nos. 6,821,505 and 7,083,784; and WO 09/058492). In addition,the half-life of antibodies of the invention may be increased byconjugation to PEG or Albumin by techniques widely utilized in the art.In some embodiments antibodies comprising Fc variant regions of theinvention have an increased half-life of about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%,about 95%, about 100%, about 125%, about 150% or more as compared to anantibody comprising a native Fc region. In some embodiments antibodiescomprising Fc variant regions have an increased half-life of about 2fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20fold, about 50 fold or more, or is between 2 fold and 10 fold, orbetween 5 fold and 25 fold, or between 15 fold and 50 fold, as comparedto an antibody comprising a native Fc region.

In one embodiment, the present invention provides Fc variants, whereinthe Fc region comprises a modification (e.g., amino acid substitutions,amino acid insertions, amino acid deletions) at one or more positionsselected from the group consisting of 221, 225, 228, 234, 235, 236, 237,238, 239, 240, 241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256,257, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296,297, 298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416,419, 421, 428, 433, 434, 435, 436, 440, and 443 as numbered by the EUindex as set forth in Kabat. Optionally, the Fc region may comprise amodification at additional and/or alternative positions known to oneskilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; 7,083,784; 7,317,091; 7,217,797; 7,276,585; 7,355,008;2002/0147311; 2004/0002587; 2005/0215768; 2007/0135620; 2007/0224188;2008/0089892; WO 94/29351; and WO 99/58572). Additional, useful aminoacid positions and specific substitutions are exemplified in Tables 2,and 6-10 of U.S. Pat. No. 6,737,056; the tables presented in FIG. 41 ofUS 2006/024298; the tables presented in FIGS. 5, 12, and 15 of US2006/235208; the tables presented in FIGS. 8, 9 and 10 of US2006/0173170 and the tables presented in FIGS. 8-10, 13 and 14 of WO09/058492.

In a specific embodiment, the present invention provides an Fc variant,wherein the Fc region comprises at least one substitution selected fromthe group consisting of 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E,234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W,235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235E, 235F, 236E,237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I,240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R. 243W, 243L 243Y, 243R,243Q, 244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S,254T, 255L, 256E, 256F, 256M, 257C, 257M, 257N, 262I, 262A, 262T, 262E,263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y,264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T,266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E,280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I,296H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 298T, 298F, 299I, 299L,299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S,320A, 320S, 322A, 322S, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V,325H, 326A, 326D, 326E, 326G, 326M, 326V, 327G, 327W, 327N, 327L, 328S,328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F,329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R,330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V,331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F,332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 333A, 333D, 333G, 333Q, 333S,333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N,378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 424F,434W, 434Y, 436H, 440Y and 443W as numbered by the EU index as set forthin Kabat. Optionally, the Fc region may comprise additional and/oralternative amino acid substitutions known to one skilled in the artincluding but not limited to those exemplified in Tables 2, and 6-10 ofU.S. Pat. No. 6,737,056; the tables presented in FIG. 41 of US2006/024298; the tables presented in FIGS. 5, 12, and 15 of US2006/235208; the tables presented in FIGS. 8, 9 and 10 of US2006/0173170 and the tables presented in FIGS. 8, 9 and 10 of WO09/058492.

In a specific embodiment, the present invention provides an Fc variantantibody, wherein the Fc region comprises at least one modification(e.g., amino acid substitutions, amino acid insertions, amino aciddeletions) at one or more positions selected from the group consistingof 228, 234, 235 and 331 as numbered by the EU index as set forth inKabat. In one embodiment, the modification is at least one substitutionselected from the group consisting of 228P, 234F, 235E, 235F, 235Y, and331S as numbered by the EU index as set forth in Kabat.

In another specific embodiment, the present invention provides an Fcvariant antibody, wherein the Fc region is an IgG4 Fc region andcomprises at least one modification at one or more positions selectedfrom the group consisting of 228 and 235 as numbered by the EU index asset forth in Kabat. In still another specific embodiment, the Fc regionis an IgG4 Fc region and the non-naturally occurring amino acids areselected from the group consisting of 228P, 235E and 235Y as numbered bythe EU index as set forth in Kabat.

In another specific embodiment, the present invention provides an Fcvariant, wherein the Fc region comprises at least one non-naturallyoccurring amino acid at one or more positions selected from the groupconsisting of 239, 330 and 332 as numbered by the EU index as set forthin Kabat. In one embodiment, the modification is at least onesubstitution selected from the group consisting of 239D, 330L, 330Y, and332E as numbered by the EU index as set forth in Kabat.

In a specific embodiment, the present invention provides an Fc variantantibody, wherein the Fc region comprises at least one non-naturallyoccurring amino acid at one or more positions selected from the groupconsisting of 252, 254, and 256 as numbered by the EU index as set forthin Kabat. In one embodiment, the modification is at least onesubstitution selected from the group consisting of 252Y, 254T and 256Eas numbered by the EU index as set forth in Kabat. In particularlypreferred antibodies of the invention, the modification is threesubstitutions 252Y, 254T and 256E as numbered by the EU index as setforth in Kabat (known as “YTE”), see U.S. Pat. No. 7,083,784.

In certain embodiments the effector functions elicited by IgG antibodiesstrongly depend on the carbohydrate moiety linked to the Fc region ofthe protein (Claudia Ferrara et al., 2006, Biotechnology andBioengineering 93:851-861). Thus, glycosylation of the Fc region can bemodified to increase or decrease effector function (see for examples,Umana et al., 1999, Nat. Biotechnol 17:176-180; Davies et al., 2001,Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. Nos. 6,602,684; 6,946,292; 7,064,191; 7,214,775; 7,393,683;7,425,446; 7,504,256; U.S. Publication. Nos. 2003/0157108; 2003/0003097;2009/0010921; Potillegent™ technology (Biowa, Inc. Princeton, N.J.);GlycoMAb™ glycosylation engineering technology (GLYCART biotechnologyAG, Zurich, Switzerland)). Accordingly, in one embodiment the Fc regionsof antibodies of the invention comprise altered glycosylation of aminoacid residues. In another embodiment, the altered glycosylation of theamino acid residues results in lowered effector function. In anotherembodiment, the altered glycosylation of the amino acid residues resultsin increased effector function. In a specific embodiment, the Fc regionhas reduced fucosylation. In another embodiment, the Fc region isafucosylated (see for examples, U.S. Patent Application Publication No.2005/0226867). In one aspect, these antibodies with increased effectorfunction, specifically ADCC, as generated in host cells (e.g., CHOcells, Lemna minor) engineered to produce highly defucosylated antibodywith over 100-fold higher ADCC compared to antibody produced by theparental cells (Mori et al., 2004, Biotechnol Bioeng 88:901-908; Cox etal., 2006, Nat Biotechnol., 24:1591-7).

Addition of sialic acid to the oligosaccharides on IgG molecules canenhance their anti-inflammatory activity and alters their cytotoxicity(Keneko et al., Science, 2006, 313:670-673; Scallon et al., Mol. Immuno.2007 March; 44(7):1524-34). The studies referenced above demonstratethat IgG molecules with increased sialylation have anti-inflammatoryproperties whereas IgG molecules with reduced sialylation have increasedimmunostimulatory properties (e.g., increase ADCC activity). Therefore,an antibody can be modified with an appropriate sialylation profile fora particular therapeutic application (US Publication No. 2009/0004179and International Publication No. WO 2007/005786).

In one embodiment, the Fc regions of antibodies of the inventioncomprise an altered sialylation profile compared to the native Fcregion. In one embodiment, the Fc regions of antibodies of the inventioncomprise an increased sialylation profile compared to the native Fcregion. In another embodiment, the Fc regions of antibodies of theinvention comprise a decreased sialylation profile compared to thenative Fc region.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Ghetieet al., 1997, Nat Biotech. 15:637-40; Duncan et al., 1988, Nature332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al.,1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA92:11980-11984; Jefferis et al., 1995, Immunol Lett. 44:111-117; Lund etal., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett54:101-104; Lund et al., 1996, J Immunol 157:4963-4969; Armour et al.,1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000, J Immunol164:4178-4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xu et al.,2000, Cell Immunol 200:16-26; Idusogie et al., 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 7,122,637; 7,183,387; 7,332,581;7,335,742; 7,371,826; 6,821,505; 6,180,377; 7,317,091; 7,355,008;2004/0002587; and WO 99/58572. Other modifications and/or substitutionsand/or additions and/or deletions of the Fc domain will be readilyapparent to one skilled in the art.

Glycosylation

In addition to the ability of glycosylation to alter the effectorfunction of antibodies, modified glycosylation in the variable regioncan alter the affinity of the antibody for antigen. In one embodiment,the glycosylation pattern in the variable region of the presentantibodies is modified. For example, an aglycoslated antibody can bemade (i.e., the antibody lacks glycosylation). Glycosylation can bealtered to, for example, increase the affinity of the antibody forantigen. Such carbohydrate modifications can be accomplished by, forexample, altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the antibody forantigen. Such an approach is described in further detail in U.S. Pat.Nos. 5,714,350 and 6,350,861. One or more amino acid substitutions canalso be made that result in elimination of a glycosylation site presentin the Fc region (e.g., Asparagine 297 of IgG). Furthermore,aglycosylated antibodies may be produced in bacterial cells which lackthe necessary glycosylation machinery.

Antibody Conjugates

In certain embodiments, the antibodies of the invention are conjugatedor covalently attached to a substance using methods well known in theart. In one embodiment, the attached substance is a therapeutic agent, adetectable label (also referred to herein as a reporter molecule) or asolid support. Suitable substances for attachment to antibodies include,but are not limited to, an amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a drug, a hormone, a lipid, a lipid assembly, asynthetic polymer, a polymeric microparticle, a biological cell, avirus, a fluorophore, a chromophore, a dye, a toxin, a hapten, anenzyme, an antibody, an antibody fragment, a radioisotope, solidmatrixes, semi-solid matrixes and combinations thereof. Methods forconjugation or covalently attaching another substance to an antibody arewell known in the art.

In certain embodiments, the antibodies of the invention are conjugatedto a solid support. Antibodies may be conjugated to a solid support aspart of the screening and/or purification and/or manufacturing process.Alternatively antibodies of the invention may be conjugated to a solidsupport as part of a diagnostic method or composition. A solid supportsuitable for use in the present invention is typically substantiallyinsoluble in liquid phases. A large number of supports are available andare known to one of ordinary skill in the art. Thus, solid supportsinclude solid and semi-solid matrixes, such as aerogels and hydrogels,resins, beads, biochips (including thin film coated biochips),microfluidic chip, a silicon chip, multi-well plates (also referred toas microtitre plates or microplates), membranes, conducting andnon-conducting metals, glass (including microscope slides) and magneticsupports. More specific examples of solid supports include silica gels,polymeric membranes, particles, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a reactive functionalgroup, including, but not limited to, hydroxyl, carboxyl, amino, thiol,aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate,isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc., forattaching the antibodies of the invention.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the antibodies of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

In certain embodiments, the antibodies of the invention are conjugatedto labels for purposes of diagnostics and other assays wherein theantibody and/or its associated ligand may be detected. A labelconjugated to an antibody and used in the present methods andcompositions described herein, is any chemical moiety, organic orinorganic, that exhibits an absorption maximum at wavelengths greaterthan 280 nm, and retains its spectral properties when covalentlyattached to an antibody. Labels include, without limitation, achromophore, a fluorophore, a fluorescent protein, a phosphorescent dye,a tandem dye, a particle, a hapten, an enzyme and a radioisotope.

In certain embodiments, the antibodies are conjugated to a fluorophore.As such, fluorophores used to label antibodies of the invention include,without limitation; a pyrene (including any of the correspondingderivative compounds disclosed in U.S. Pat. No. 5,132,432), ananthracene, a naphthalene, an acridine, a stilbene, an indole orbenzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1, 3-diazole (NBD), a cyanine (including anycorresponding compounds in U.S. Pat. Nos. 6,977,305 and 6,974,873), acarbocyanine (including any corresponding compounds in U.S. Ser. No.09/557,275; U.S.; U.S. Pat. Nos. 4,981,977; 5,268,486; 5,569,587;5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003;6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; andpublications WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1065 250 A1), a carbostyryl, a porphyrin, a salicylate, an anthranilate,an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene(including any corresponding compounds disclosed in U.S. Pat. Nos.4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene(including any corresponding compounds disclosed in U.S. Pat. Nos.6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343; 5,227,487;5,442,045; 5,798,276; 5,846,737; 4,945,171; U.S. Ser. Nos. 09/129,015and 09/922,333), an oxazine (including any corresponding compoundsdisclosed in U.S. Pat. No. 4,714,763) or a benzoxazine, a carbazine(including any corresponding compounds disclosed in U.S. Pat. No.4,810,636), a phenalenone, a coumarin (including an correspondingcompounds disclosed in U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980and 5,830,912), a benzofuran (including an corresponding compoundsdisclosed in U.S. Pat. Nos. 4,603,209 and 4,849,362) and benzphenalenone(including any corresponding compounds disclosed in U.S. Pat. No.4,812,409) and derivatives thereof. As used herein, oxazines includeresorufins (including any corresponding compounds disclosed in U.S. Pat.No. 5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

In a specific embodiment, the fluorophores conjugated to the antibodiesdescribed herein include xanthene (rhodol, rhodamine, fluorescein andderivatives thereof) coumarin, cyanine, pyrene, oxazine andborapolyazaindacene. In other embodiments, such fluorophores aresulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins,fluorinated coumarins and sulfonated cyanines. Also included are dyessold under the tradenames, and generally known as, ALEXA FLUOR®,DyLight, CY® Dyes, BODIPY®, OREGON GREEN®, PACIFIC BLUE™, IRDYE®, FAM,FITC, and ROX™.

The choice of the fluorophore attached to the antibody will determinethe absorption and fluorescence emission properties of the conjugatedantibody. Physical properties of a fluorophore label that can be usedfor antibody and antibody bound ligands include, but are not limited to,spectral characteristics (absorption, emission and stokes shift),fluorescence intensity, lifetime, polarization and photo-bleaching rate,or combination thereof. All of these physical properties can be used todistinguish one fluorophore from another, and thereby allow formultiplexed analysis. In certain embodiments, the fluorophore has anabsorption maximum at wavelengths greater than 480 nm. In otherembodiments, the fluorophore absorbs at or near 488 nm to 514 nm(particularly suitable for excitation by the output of the argon-ionlaser excitation source) or near 546 nm (particularly suitable forexcitation by a mercury arc lamp). In other embodiment a fluorophore canemit in the NIR (near infra red region) for tissue or whole organismapplications. Other desirable properties of the fluorescent label mayinclude cell permeability and low toxicity, for example if labeling ofthe antibody is to be performed in a cell or an organism (e.g., a livinganimal).

In certain embodiments, an enzyme is a label and is conjugated to anantibody described herein. Enzymes are desirable labels becauseamplification of the detectable signal can be obtained resulting inincreased assay sensitivity. The enzyme itself does not produce adetectable response but functions to break down a substrate when it iscontacted by an appropriate substrate such that the converted substrateproduces a fluorescent, colorimetric or luminescent signal. Enzymesamplify the detectable signal because one enzyme on a labeling reagentcan result in multiple substrates being converted to a detectablesignal. The enzyme substrate is selected to yield the preferredmeasurable product, e.g. colorimetric, fluorescent or chemiluminescence.Such substrates are extensively used in the art and are well known byone skilled in the art.

In one embodiment, colorimetric or fluorogenic substrate and enzymecombination uses oxidoreductases such as horseradish peroxidase and asubstrate such as 3,3′-diaminobenzidine (DAB) and3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color(brown and red, respectively). Other colorimetric oxidoreductasesubstrates that yield detectable products include, but are not limitedto: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates include, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplex® Red reagent and its variants (U.S.Pat. No. 4,384,042) and reduced dihydroxanthenes, includingdihydrofluoresceins (U.S. Pat. No. 6,162,931) and dihydrorhodaminesincluding dihydrorhodamine 123. Peroxidase substrates that are tyramides(U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a uniqueclass of peroxidase substrates in that they can be intrinsicallydetectable before action of the enzyme but are “fixed in place” by theaction of a peroxidase in the process described as tyramide signalamplification (TSA). These substrates are extensively utilized to labelantigen in samples that are cells, tissues or arrays for theirsubsequent detection by microscopy, flow cytometry, optical scanning andfluorometry.

In another embodiment, a colorimetric (and in some cases fluorogenic)substrate and enzyme combination uses a phosphatase enzyme such as anacid phosphatase, an alkaline phosphatase or a recombinant version ofsuch a phosphatase in combination with a colorimetric substrate such as5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate,resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates(U.S. Pat. Nos. 5,316,906 and 5,443,986).

Glycosidases, in particular beta-galactosidase, beta-glucuronidase andbeta-glucosidase, are additional suitable enzymes. Appropriatecolorimetric substrates include, but are not limited to,5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similarindolyl galactosides, glucosides, and glucuronides, o-nitrophenylbeta-D-galactopyranoside (ONPG) and p-nitrophenylbeta-D-galactopyranoside. In one embodiment, fluorogenic substratesinclude resoruf in beta-D-galactopyranoside, fluorescein digalactoside(FDG), fluorescein diglucuronide and their structural variants (U.S.Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236),4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferylbeta-D-galactopyranoside and fluorinated coumarinbeta-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes include, but are not limited to, hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases, and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare preferred for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful.

In another embodiment, haptens such as biotin, are also utilized aslabels. Biotin is useful because it can function in an enzyme system tofurther amplify the detectable signal, and it can function as a tag tobe used in affinity chromatography for isolation purposes. For detectionpurposes, an enzyme conjugate that has affinity for biotin is used, suchas avidin-HRP. Subsequently a peroxidase substrate is added to produce adetectable signal.

Haptens also include hormones, naturally occurring and synthetic drugs,pollutants, allergens, affector molecules, growth factors, chemokines,cytokines, lymphokines, amino acids, peptides, chemical intermediates,nucleotides and the like.

In certain embodiments, fluorescent proteins may be conjugated to theantibodies as a label. Examples of fluorescent proteins include greenfluorescent protein (GFP) and the phycobiliproteins and the derivativesthereof. The fluorescent proteins, especially phycobiliprotein, areparticularly useful for creating tandem dye labeled labeling reagents.These tandem dyes comprise a fluorescent protein and a fluorophore forthe purposes of obtaining a larger stokes shift wherein the emissionspectra is farther shifted from the wavelength of the fluorescentprotein's absorption spectra. This is particularly advantageous fordetecting a low quantity of antigen in a sample wherein the emittedfluorescent light is maximally optimized, in other words little to noneof the emitted light is reabsorbed by the fluorescent protein. For thisto work, the fluorescent protein and fluorophore function as an energytransfer pair wherein the fluorescent protein emits at the wavelengththat the fluorophore absorbs at and the fluorphore then emits at awavelength farther from the fluorescent proteins than could have beenobtained with only the fluorescent protein. A particularly usefulcombination is the phycobiliproteins disclosed in U.S. Pat. Nos.4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophoresdisclosed in U.S. Pat. No. 5,798,276, or the sulfonated cyaninefluorophores disclosed in U.S. Pat. Nos. 6,977,305 and 6,974,873; or thesulfonated xanthene derivatives disclosed in U.S. Pat. No. 6,130,101 andthose combinations disclosed in U.S. Pat. No. 4,542,104. Alternatively,the fluorophore functions as the energy donor and the fluorescentprotein is the energy acceptor.

In certain embodiments, the label is a radioactive isotope. Examples ofsuitable radioactive materials include, but are not limited to, iodine(¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹¹In, ¹¹²In, ^(113m)IN, ^(115m)IN), technetium (⁹⁹Tc,^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd)molybdenum (⁹⁹Mo), xenon (¹³⁵Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and⁹⁷Ru.

Medical Treatments and Uses

The antibodies and binding fragments thereof of the invention andvariants thereof may be used for the treatment of influenza A virusinfection, for the prevention of influenza A virus infection and/or forthe detection, diagnosis and/or prognosis of influenza A virusinfection.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be tissue samples taken from,for example, nasal passages, sinus cavities, salivary glands, lung,liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart,ovaries, pituitary, adrenals, thyroid, brain or skin. The methods ofdetection, diagnosis, and/or prognosis may also include the detection ofan antigen/antibody complex.

In one embodiment, the invention provides a method of treating a subjectby administering to the subject an effective amount of an antibody or anbinding fragment thereof, according to the invention, or apharmaceutical composition that includes the antibody or bindingfragment thereof. In one embodiment, the antibody or binding fragmentthereof is substantially purified (i.e., substantially free fromsubstances that limit its effect or produce undesired side-effects). Inone embodiment, the antibody or binding fragment thereof of theinvention is administered post-exposure, or after the subject has beenexposed to influenza A virus or is infected with influenza A virus. Inanother embodiment, the antibody or binding fragment thereof of theinvention is administered pre-exposure, or to a subject that has not yetbeen exposed to influenza A virus or is not yet infected with influenzaA virus. In one embodiment, the antibody or binding fragment thereof ofthe invention is administered to a subject that is sero-negative for oneor more influenza A subtypes. In another embodiment, the antibody orbinding fragment thereof of the invention is administered to a subjectthat is sero-positive for one or more influenza A subtypes. In oneembodiment, the antibody or binding fragment thereof of the invention isadministered to a subject within 1, 2, 3, 4, 5 days of infection orsymptom onset. In another embodiment, the antibody or binding fragmentthereof of the invention can be administered to a subject after 1, 2, 3,4, 5, 6, or 7 days, and within 2, 3, 4, 5, 6, 7, 8, 9 or 10 days afterinfection or symptom onset.

In one embodiment, the method reduces influenza A virus infection in thesubject. In another embodiment, the method prevents, reduces the risk ordelays influenza A virus infection in the subject. In one embodiment,the subject is a mammal. In a more particular embodiment, the subject ishuman. In one embodiment, the subject includes, but is not limited to,one who is particularly at risk of or susceptible to influenza A virusinfection, including, for example, an immunocompromised subject.

Treatment can be a single dose schedule or a multiple dose schedule andthe antibody or binding fragment thereof of the invention can be used inpassive immunization.

In one embodiment, the antibody or binding fragment thereof of theinvention is administered to a subject in combination with one or moreantiviral medications. In one embodiment, the antibody or bindingfragment thereof of the invention is administered to a subject incombination with one or more small molecule antiviral medications. Smallmolecule antiviral medications include neuraminidase inhibitors such asoseltamivir (TAMIFLU®), zanamivir (RELENZA®) and adamantanes such asAmantadine and rimantadine.

In another embodiment, the invention provides a composition for use as amedicament for the prevention or treatment of an influenza A virusinfection. In another embodiment, the invention provides the use of anantibody or binding fragment thereof of the invention and/or a proteincomprising an epitope to which an antibody or binding fragment thereofof the invention binds in the manufacture of a medicament for treatmentof a subject and/or diagnosis in a subject.

Antibodies and fragments thereof as described in the present inventionmay also be used in a kit for the diagnosis of influenza A virusinfection. Further, epitopes capable of binding an antibody of theinvention may be used in a kit for monitoring the efficacy ofvaccination procedures by detecting the presence of protectiveanti-influenza A virus antibodies. Antibodies, antibody fragment, orvariants and derivatives thereof, as described in the present inventionmay also be used in a kit for monitoring vaccine manufacture with thedesired immunogenicity.

The invention also provides a method of preparing a pharmaceuticalcomposition, which includes the step of admixing a monoclonal antibodywith one or more pharmaceutically-acceptable carriers, wherein theantibody is a monoclonal antibody according to the invention describedherein.

Various delivery systems are known and can be used to administer theantibody or binding fragment thereof of the invention, including, butnot limited to, encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the antibody orantibody fragment, receptor-mediated endocytosis, construction of anucleic acid as part of a retroviral or other vector, delivery of nakednucleotide acids by electroporation delivery technology (as described inMuthumani et al., PLoS One. 2013 Dec. 31; 8(12):e84234. doi:10.1371/journal.pone.0084234. eCollection 2013) etc. Methods ofintroduction include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucutaneous linings (e.g., oral mucosa,rectal and intestinal mucosa, etc.) and may be administered togetherwith other biologically active agents, including, but not limited tosmall molecule antiviral compositions. Administration can be systemic orlocal. Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. In yetanother embodiment, the composition can be delivered in a controlledrelease system.

The present invention also provides pharmaceutical compositions. Suchcompositions include a therapeutically effective amount of an antibodyor binding fragment thereof of the invention, and a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” as usedherein, means approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. In one embodiment, the pharmaceuticalcomposition contains a therapeutically effective amount of the antibodyor binding fragment thereof, preferably in purified form, together witha suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

Typically, for antibody therapeutics, the dosage administered to apatient is between about 0.1 mg/kg to 100 mg/kg of the patient's bodyweight.

LIST OF FIGURES

FIG. 1A shows the binding of Antibody 3 and Antibody 12 to surfaceexpressed HA protein of subtypes H11, H12, H13, H16, H17, H4, H10, H14,and H15. Histograms depict the number of cells vs the florescenceintensity of antibody binding to HA transfected cells in white or mocktransfected cells in grey. FIG. 1B shows the percent inhibition of lowpH induced fusion of chicken red blood cells and A/Puerto Rico/8/34 inthe presence of Antibody 3, Antibody 12, or MPE8v3 (non-relevant viralfusion protein antibody) (Corti D et al., 2013, Nature 501). FIGS. 1Cand 1D show immunoblots of uncleaved (HA0), recombinant H1 HA afterdigestion with trypsin for 5, 10 or 20 minutes. Digestion reactionscontained either HA alone (input), or HA pre-treated with FI6v4(disclosed in WO2013/011347A1), Antibody 3, FE17.23 (globular headspecific mAb) (Corti D et al., 2010, J Clin Invest 120) or non-relevantcontrol antibody (Ctrl. IgG) in 10, and HA alone (input), or HApre-treated with Antibody 3, Antibody 12, Antibody 14, or non-relevantcontrol antibody (Ctrl. IgG) in 1D.

FIG. 2 shows the percentage of NK cell mediated killing of A/HK/8/68infected MDCK cells in the presence of increasing amount of Antibody 3,Antibody 11, Antibody 12, and Antibody 14.

FIG. 3 shows the percentage of macrophages that phagocytosed A/HK/8/68HA expressing MDCK target cells in the presence of increasing amount ofAntibody 3, Antibody 11, Antibody 12, and Antibody 14, or non-relevantisotype control (Ctrl. IgG).

FIG. 4 shows the percentage of complement dependent killing of A/PR/8/34infected MDCK cells in the presence of increasing amount of Antibody 3,Antibody 11, Antibody 12, and Antibody 14.

FIG. 5 shows the percentage of surviving animals in each group of astudy when different concentrations of Antibody 3 or a non-relevantcontrol antibody (Ctrl. IgG) were administered to mice 4 hours beforeinfection with a lethal dose of H1N1 influenza virus.

FIG. 6 shows the percentage of surviving animals in each group of astudy in which different concentrations of Antibody 3 or a non-relevantcontrol antibody (Ctrl. IgG) were administered to mice 4 hours beforeinfection with a lethal dose of H3 influenza virus.

FIG. 7 shows the percentage of surviving animals in each group when micewere infected with a lethal dose of H1N1 influenza virus and treated atdifferent time points (1 and 2 days post-infection) with 30 mg/kg ofAntibody 3 or a non-relevant control antibody (Ctrl. IgG).

FIG. 8 shows the percentage of surviving animals in each group of astudy in which mice were infected with a lethal dose of H3 influenzavirus and treated at different time points (3, 4 and 5 dayspost-infection) with 30 mg/kg of Antibody 3 or a non-relevant controlantibody (Ctrl. IgG).

FIG. 9 shows the percentage of surviving animals in each group of astudy in which mice were infected with a lethal dose of H1N1 influenzavirus and treated at 1 day post infection with 2 mg/kg of Antibody 3,Antibody 11, Antibody 12, Antibody 14 or a non-relevant control antibody(Ctrl. IgG).

FIG. 10 shows the percentage of surviving animals in each group of astudy in which mice were infected with a lethal dose of H3 influenzavirus and treated at 2 day post infection with 3 mg/kg of Antibody 3,Antibody 11, Antibody 12, Antibody 14 or a non-relevant control antibody(Ctrl. IgG).

FIG. 11 shows the percentage of surviving animals in each group of astudy in which mice were infected with a lethal dose of H1N1 influenzavirus and treatment of 25 mg/kg BID oseltamivir for 5 days, 10 mg/kg ofAntibody 12, or 10 mg/kg of non-relevant control antibody (Ctrl. IgG)was initiated at different time points (4 hr before, 1 day post, or 2days post infection).

FIG. 12 shows the percentage of surviving animals in each group of astudy in which mice were infected with a lethal dose of H3 influenzavirus and treatment of 25 mg/kg oraloseltamivir twice daily (BID) for 5days, or single dose 10 mg/kg of Antibody 12, or 10 mg/kg of anon-relevant control antibody (Ctrl. IgG) that was initiated at varioustime points (1, 2, 3 or 4 days post infection).

FIG. 13 shows the percentage of surviving animals in each group in astudy that mice were infected with a lethal dose of H3 influenza virusand treated with Antibody 12 at 2.5 mg/kg or 0.3 mg/kg single dose,oseltamivir at 25 mg/kg BID for 5 days, or a combination of Antibody 12at 2.5 mg/kg or 0.3 mg/kg and oseltamivir at 25 mg/kg BID for 5 days at2 days post infection.

FIG. 14 shows the percentage of surviving ferrets in each group of astudy after infection with a lethal dose of H5N1 influenza virus andtreatment with 25 mg/kg single dose Antibody 12, 25 mg/kg BIDoseltamivir for 5 days, or a non-relevant control antibody (Ctrl. IgG)at 1, 2, or 3 days post infection.

FIG. 15 shows an alignment of HA2 Protein of Influenza A Strains Used inMARM selection.

FIG. 16 shows the VH percent identity of anti-HA Antibodies 1-15 andAntibody 3-GL.

FIG. 17 shows the VH alignment of anti-HA Antibodies 1-15 and Antibody3-GL.

FIG. 18 shows the VL percent identity of anti-HA Antibodies 1-15 andAntibody 3-GL.

FIG. 19 shows the VL alignment of anti-HA Antibodies 1-15 and Antibody3-GL.

EXAMPLES Example 1 Construction and Optimization of Human MonoclonalAntibodies Isolated from Memory B Cells

The CD22+ IgG+ B cells were sorted from cryopreserved peripheral bloodmononuclear cells (PBMCs) of a donor selected for high titers ofheterosubtypic antibodies and immortalized at 3 cells/well using EpsteinBarr Virus (EBV) and CpG oligodeoxynucleotide 2006 and feeder cells.Culture supernatants containing antibodies were harvested after 14 daysand screened by ELISA binding assay to determine the binding activityagainst H5 (A/Vietnam/1203/04) and H7 (A/NLD/03) hemagglutinin (HA),respectively. Four B cell clones (Antibody 1, Antibody 4, Antibody 7,and Antibody 9) were found to bind specifically to both HAs and weretherefore collected. The VH and VL genes of these clones were sequencedand found to be clonally related according to the homology analysisperformed on VH and VL V, D and J fragments using the Kabat database. Ofnote, the VH of Antibody 4 was found to have a degenerate nucleotidesite in the HCDR3 encoding for either valine (encoded in Antibody 5) orglutamate (encoded in Antibody 6). The VH and VL genes of the fourantibodies were cloned into IgG1 expression vectors (minor sequencemodifications to facilitate cloning and or codon optimization resultedin the five antibodies; Antibody 3, Antibody 5, Antibody 6, Antibody 8and Antibody 10; used in the following Examples) and recombinantantibodies were produced by transient transfection of mammalian celllines derived from HEK or CHO cells. Supernatants from transfected cellswere collected after 7-10 days of culture, and IgGs were affinitypurified by Protein A chromatography, and dialyzed into PBS. Antibody 3was further optimized to create variants in which non-germline encodedsomatic mutations located in the framework regions were changed to thegermline encoded amino acid, and the CDR regions were subjected toparsimonious mutagenesis. Full IgG constructs containing differentmutations were expressed as described above and the crude supernatantswere screened by ELISA to select clones that had increased bindingactivity to H3 and H1 HA proteins. ELISA was performed using a coatingconcentration of 0.15 μg/ml of rabbit anti-human IgG in order to captureand normalize IgG from the supernatants, and then 0.5 μg/ml ofbiotinylated HA subtype H1 (A/California/7/04 (H1N1)) or subtype H3(A/Perth/16/09 (H3N2)) was added and incubated for one hour. Binding wasdetected by the addition of streptavidin-HRP (1:5000), and developmentabsorbance was read at 450 nm. The beneficial single mutationsconferring better binding were combined and cloned into a combinatoriallibrary, which were expressed and screened by ELISA as described above.This library approach resulted in the creation of 5 additional Antibody3 variants that were further characterized (Antibodies 11-15).

Example 2 Anti-HA Neutralizing Antibody (nAb) Binds to HA of DifferentSubtypes

To test if the epitope of the anti-HA antibodies is conserved among HAsof different subtypes, a HA cross-reactivity ELISA binding assay wasperformed. A 384-well Maxisorb ELISA plate (Nunc) was coated overnightat 4° C. with 0.5 ug/ml recombinant HA (rHA), subtype H1(A/California/7/09 (H1N1)), subtype H2 (A/Swine/MO/06 (H2N3)), subtypeH3 (A/Perth/16/09 (H3N2)), subtype H5 (A/Vietnam/1203/04(H5N1)), subtypeH6 (A/teal/HK/W312/97(H6N1)), subtype H7 (A/Netherlands/219/03(H7N7))and subtype H9 (A/chicken/HK/G9/97(H9N2)) in PBS. The plate was washedwith PBS containing 0.1% v/v Tween-20 to remove uncoated protein andsubsequently blocking solution containing 1% (w/v) casein (ThermoScientific) was added and incubated for 1 hr at room temperature. Theblocking solution was discarded and 3-fold serially diluted anti-HAantibodies in blocking solution (Casein-PBS (Thermo Scientific) wereadded and incubated for 1 hr at room temperature. The plate was washedthree times and bound antibodies were detected using aperoxidase-conjugated mouse anti-human IgG antibody (Jackson). Thebinding activity of antibody was calculated by either measuring thechemiluminescent signal after addition of Supersignal Pico substrate(Thermo Scientific) or by measuring the color change at 450 nm afterincubation with Tetramethylbenzidine (TMB) one component substrate (KPL)followed by the addition of 2N sulfuric acid to stop the reaction.

TABLE 1 Binding to rHA by ELISA (EC₅₀, ug/ml) H1 H2 H5 H6 H9 H3 H7A/CA/7/09 A/swine/MO/06 A/VN/1203/04 A/HK/W312/97 A/HK/G9/97A/Perth/16/09 A/NLD/219/03 Antibody 3 0.026 0.028 0.022 0.043 0.0120.019 0.020 Antibody 5 0.045 0.048 0.041 0.047 >6 0.030 0.024 Antibody 60.311 0.213 0.256 0.214 >6 0.064 0.116 Antibody 8 0.069 0.058 0.0440.091 >6 0.067 0.015 Antibody 10 0.073 0.075 0.058 0.097 2.699 0.0490.034

Table 1 shows that all anti-HA IgGs tested bound to recombinant HA ofsubtypes H1, H2, H3, H5, H6, H9 and H7. Recombinant HA of subtype H9 wasrecognized by Antibody 3 and Antibody 10, but not by Antibody 5,Antibody 6 and Antibody 8 at the highest concentration of antibodytested (6 ug/ml). This indicates that the epitopes of the majority ofthese anti-HA IgGs are conserved among HA molecules of differentsubtypes.

TABLE 2 Binding to rHA by ELISA (EC₅₀, ug/ml) H1 H2 H5 H6 H9 H3 H7A/CA/7/09 A/swine/MO/06 A/VN/1203/04 A/HK/W312/97 A/HK/G9/97A/Perth/16/09 A/NLD/219/03 Antibody 3 0.045 0.095 0.099 0.072 0.1710.129 0.258 Antibody 11 0.085 0.126 0.168 0.129 0.164 0.176 0.553Antibody 12 0.059 0.088 0.084 0.083 0.098 0.028 0.061 Antibody 13 0.0500.062 0.080 0.097 0.161 0.023 0.049 Antibody 14 0.048 0.079 0.061 0.0730.095 0.030 0.064 Antibody 15 0.028 0.042 0.035 0.043 0.065 0.032 0.035

Table 2 shows that all anti-HA IgGs variants tested bound to recombinantHA of group 1 subtypes H1, H2, H5, H6 and H9 with similar EC₅₀ values.All the variants bound to group 2 HA proteins (H3 and H7), however,Antibody 11 and Antibody 3 showed decreased activity with increased EC₅₀values compared to the Antibodies 12-15.

To extend these binding results to include more diverse HA subtypes, weperformed additional binding studies using a flow cytometry basedbinding to HA transfected cells. In this assay, HEK cells weretransiently transfected with full-length wild type HA expressingplasmids of subtype H4 A/duck/Czechoslovakia/56 (H4N6)), subtype H10(A/chicken/Germany/N49 (H10N7)), subtype H11 (A/duck/Memphis/546/74(H11N9)), subtype H12 (A/duck/Alberta/60/76 (H12N5)), subtype H13(A/gull/Maryland/704/77 (H13N6)), subtype H14(A/mallard/Astrakhan/263/82 (H14N5)), subtype H15 (A/shearwater/WestAustralia/2576/79 (H15N9)), subtype H16 (A/black-headed gull/Sweden/2/99(H16N3)), and subtype H17 (A/little yellow-shoulderedbat/Guatemala/164/2009 (H17N10)). Forty-eight hours after transfection,cells were detached with trypsin, and incubated with 5 ug/ml of Antibody3 or Antibody 12 on ice for 1 hour. After the hour incubation, theantibody bound to cell-surface expressed HA protein was then stainedwith a goat anti-human IgG Daylight 649 (Jackson ImmunoResearch), andwhich was detected by flow cytometry. FIG. 1A shows the shift influorescence intensity when antibody is bound to HA expressing cells(white) vs mock-transfected cells (grey) from each of the subtypes.Antibody 3 bound to all HAs tested with the exception of H12, whileAntibody 12 bound to all HAs tested from both groups (group 1 H11, H12,H13, H16, and H17 and group 2 H4, H10, H14, and H15)

Example 3 Kinetic Characterization of the HA Binding to Antibody 3 andAntibody 5 IgG1 by Using Octet

Affinity measurements were performed using a ForteBio Octet QK 384Kinetic Analyzer (Menlo Park, Calif.) in 384 slanted-well plates. Allreagents were diluted in Octet Kinetics Buffer (ForteBio). His-tagged HAof different subtypes: subtype H1 (A/California/7/04 (H1N1)) and subtypeH3 (A/Perth/16/09 (H3N2)) were immobilized onto anti-His sensors at 10μg/mL. Anti-HA mAb association/dissociation were then monitored in2-fold dilutions from 100 nM, plus a zero mAb control.

Association and dissociation raw data were corrected for any drift inthe zero mAb controls, and then exported to GraphPad Prism (San Diego,Calif.) for affinity curve fitting. Data were fitted using globalassociation/dissociation fitting with an imposed limit of >5×10⁻⁶ sec⁻¹.As shown in Table 3, both antibodies have a very high affinity bindingto H1 at pM level with a slow dissociation rate under limit ofdetection. Similar K_(on), K_(off) and Kd of both antibodies wereobserved with H3 trimer at sub-nM level.

TABLE 3 Kinetic Binding Analysis of Pan A mAbs on rHA by Octet H1A/CA/7/09 H3 A/Perth/16/09 K_(on) K_(off) Kd K_(on) K_(off) Kd (e5M⁻¹s⁻¹) (e⁻⁶s⁻¹) (pM) (e5 M⁻¹s⁻¹) (e⁻⁶s⁻¹) (pM) Antibody 3 5.3 <5 11 3.362 188 Antibody 5 10 <5 5 2.6 88 338

Example 4 In Vitro Cross-Reactive Neutralizing Activity of Anti-HA IgG1sAgainst Virus of Different Subtypes

The microneutralization assay (MNA) was modified from a previouslydescribed accelerated viral inhibition assay using neuraminidaseactivity (NA) as a read-out (Hassantoufighi, A. et al. 2010, Vaccine28:790). Briefly, MNA were performed on MDCK cells that were cultured inMEM medium (Invitrogen) supplemented with antibiotics, glutamine(complete MEM medium) and 10% (v/v) fetal bovine serum. 60 TCID₅₀ (50%tissue culture infectious doses) of virus was added to three-folddilutions of antibody in a 384-well plate in complete MEM mediumcontaining 0.75 ug/ml Trypsin (Worthington) in duplicate wells, after 30minutes incubation at room temperature, 2×10⁴ cells/well were added tothe plate. After incubation at 33° C. 5% CO₂ incubator for approximately40 hr, the NA activity was measured by adding a fluorescently-labeledsubstrate, methylumbelliferyl-N-acetyl neuraminic acid (MU-NANA) (Sigma)to each well and incubated at 37° C. for 1 hr. Virus replicationrepresented by NA activity was quantified by reading fluorescence inFluorometer Envison (PerkinElmer) using the following settings:excitation 355 nm, emission 460 nm; 10 flashes per well. Theneutralization titer (50% inhibitory concentration [IC₅₀]) is expressedas the final antibody concentration that reduced the fluorescence signalby 50% compared to cell control wells. Table 4 and 5 showed anti-HAantibodies neutralized influenza A viruses of different subtypes testedbelow: H1-PR34 (A/Puerto Rico/8/34 (H1N1)); H1-PR34-OR (A/PuertoRico/8/34 containing the NA 274Y (N2 numbering) mutation conferringoseltamivir resistance (H1N1)); H1-FM47 (A/Fort Monmouth/1/47 (H1N1));H1-NJ76 (A/New Jersey/8/76 (H1N1)); H1-Kaw86 (A/Kawasaki/9/86 (H1N1));H1-TX91 (ca A/Texas/36/91 (H1N1)): H1-BJ95 (ca A/Beijing/262/95 (H1N1));H1-NcaI99 (ca A/New Caledonia/20/99 (H1N1)); H1-SD07 (ca A/SouthDakota/6/07 (H1N1)); H1-CA09 (ca A/California/7/09 (H1N1)); H1-CA09-OR(ca A/California/7/09 containing the NA 274Y (N2 numbering) mutationconferring oseltamivir resistance (H1N1)); H5-VN04 (ca A/Vietnam/1203/04(H5N1)); H5-HK03 (ca A/Hong Kong/213/03 (H5N1)); H9-HK97 (caA/chicken/Hong Kong/G9/97 (H9N2); H2-JP57 (ca A/Japan/57 (H2N2));H2-MO06 (ca A/swine/Missouri/06 (H2N3)); H6-HK97 (ca A/teal/HongKong/W312/97 (H6N1)); H6-AB85 (ca A/mallard/Alberta/89/85 (H6N2));H3-HK68 (A/Hong Kong/8/68 (H3N2)); H3-Vic75 (A/Victoria/3/75 (H3N2));H3-LA87 (A/Los Angeles/7/09 (H3N2)); H3-SD93 (A/Shan dong/9/93 (H3N2));H3-WH95 (ca A/Wuhan/359/95 (H3N2)); H3-Syd97 (ca A/Sydney/5/97 (H3N2));H3-WH95-OR (ca A/Wuhan/359/95 containing the NA 274Y (N2 numbering)mutation conferring oseltamivir resistance (H3N2)); H3-Pa99 (caA/Panama/2007/99 (H3N2)); H3-Wy03 (A/Wyoming/03/03 (H3N2)); H3-W105(A/Wisconsin/67/05 (H3N2)); H3-Perth09 (ca A/Perth/16/09 (H3N2)),H3-VC11 (A/Victoria/361/11 (H3N2)); H7-NLD03 (ca A/Netherlands/219/03(H7N7)); H7-BC04 (ca A/Brit. Columbia/CN-6/04 (H7N3-LP); H7-ANU13 (caA/Anhui/1/13 (H7N9).

TABLE 4 Neutralization of infectious viruses (IC₅₀ ug/ml) AntibodyAntibody Antibody Antibody Antibody Virus 3 5 6 8 10 Group 1 H1-PR341.07 1.13 4.37 3.02 2.15 H1-FM47 0.92 0.86 3.04 1.37 1.11 H1-NJ76 1.411.64 2.60 2.26 0.15 H1-Kaw86 0.58 1.01 3.51 2.11 1.62 H1-TX91 0.60 0.762.20 0.70 0.48 H1-BJ95 3.41 5.06 20.86 10.60 4.46 H1-Ncal99 0.79 0.853.00 2.06 1.26 H1-SD07 0.97 1.61 6.27 2.62 1.37 H1-CA09 2.19 2.52 5.564.50 1.62 H2-MO06 2.27 2.38 2.90 2.62 1.04 H5-VM04 2.11 2.60 8.87 3.902.21 H5-HK03 4.64 1.18 10.45 1.82 1.60 H6-HK97 1.77 2.27 3.23 2.97 1.05H9-HK97 1.79 2.43 16.47 26.39 1.76 Group 2 H3-HK68 0.68 0.39 2.04 2.820.85 H3-Vic75 0.75 0.57 1.09 3.83 0.91 H3-LA87 4.19 3.54 12.60 >50 4.59H3-SD93 9.39 6.92 19.50 >50 11.65 H3-WH95 3.96 3.72 10.54 >50 8.70H3-Syd97 3.75 3.03 6.54 >50 9.29 H3-Pa99 17.74 16.74 25.82 >50 18.71H3-Wy03 0.63 0.77 4.70 >50 1.52 H3-WI05 2.44 2.83 6.76 >50 4.46H3-Perth09 1.49 2.22 5.03 >50 2.56 H7-NLD03 4.78 4.14 >50 12.75 3.80H7-BC04 4.72 5.35 >50 14.69 3.59

Table 4 shows that anti-HA antibodies neutralize all group 1 influenza Aviruses tested. All anti-HA antibodies except Antibody 8 demonstratedneutralizing activity against all H3 influenza A viruses tested and allanti-HA antibodies except Antibody 6 exhibited neutralizing activityagainst H7-NLD03 (ca A/Netherlands/219/03 (H7N7)); H7-BC04 (ca A/Brit.Columbia/CN-6/04 (H7N3-LP).

TABLE 5 Neutralization of infectious viruses (IC₅₀ ug/ml) AntibodyAntibody Antibody Antibody Antibody Antibody Virus 3 11 12 13 14 15Group 1 H1-PR34 2.17 0.88 1.07 1.30 1.25 1.47 H1-PR34-OR 1.39 0.73 0.690.88 0.83 0.90 H1-FM47 1.04 0.43 0.28 0.50 0.44 0.35 H1-NJ76 0.57 0.130.12 0.12 0.11 0.25 H1-Kaw86 1.01 0.53 0.28 0.41 0.35 0.48 H1-TX91 0.920.11 0.12 0.09 0.09 0.13 H1-BJ95 2.98 1.01 1.31 1.86 2.09 1.81 H1-Ncal991.16 0.66 0.61 0.77 0.67 0.79 H1-SD07 2.04 0.98 0.78 1.35 1.05 0.81H1-CA09 2.07 0.90 0.98 1.23 1.07 1.17 H1-CA09-OR 2.10 0.87 0.84 1.051.23 1.35 H1-BS10 2.16 1.15 1.25 1.23 1.93 1.89 H2-JP57 0.46 0.31 0.350.47 0.67 0.33 H2-MO06 1.09 0.60 0.53 0.57 0.65 0.83 H5-VM04 1.19 0.570.31 0.56 0.33 0.28 H5-HK03 0.71 0.21 0.17 0.17 0.21 0.05 H6-AB85 0.690.24 0.32 0.29 0.26 0.19 H6-HK97 0.63 0.40 0.45 0.55 0.26 0.33 H9-HK971.18 0.36 0.31 0.29 0.44 0.35 Group 2 H3-HK68 1.37 0.46 0.42 0.44 0.650.50 H3-Vic75 1.12 0.46 0.32 0.43 0.44 0.35 H3-LA87 2.04 0.80 0.82 1.000.83 0.83 H3-SD93 3.57 1.11 1.32 1.56 1.57 1.43 H3-WH95 5.63 2.45 2.092.77 2.77 3.32 H3-WH95-OR 7.70 2.26 2.34 3.01 3.09 3.48 H3-Syd97 6.501.53 1.56 2.18 1.82 1.79 H3-Pa99 9.00 2.18 2.04 2.62 4.36 3.39 H3-WI052.62 1.07 1.09 1.19 1.19 1.30 H3-Perth09 1.30 0.17 0.25 0.28 0.47 0.50H3-VC11 3.40 0.85 0.83 1.03 1.15 1.29 H7-NLD03 4.74 0.94 0.83 2.45 1.161.30 H7-BC04 2.95 0.71 0.78 0.96 0.86 1.25 H7-ANU13 4.26 nd 2.56 nd 2.12nd

Table 5 shows that the Antibody variants (Antibodies 11-15) are moreeffective than parental Antibody 3 in neutralizing all group 1 and group2 influenza A viruses tested with decreased IC₅₀ values. In addition,antibodies also neutralized 3 viruses which have a mutation engineeredinto the NA protein conferring oseltamivir resistance (OR).

Example 5 Neutralizing Activity of Anti-HA IgGs Against Swine OriginH3N2 Viruses

The neutralizing activity of anti-HA Antibody 3 and variants (Antibodies11-15) against newly emerged swine-origin H3N2 viruses(A/Minnesota/11/2010 and A/Indiana/10/2011) was measured in amicroneutralization assay as described in Example 4. Antibody FI6v4(described in WO2013/011347A1) was used as a control antibody. As shownin Table 6 from two independent experiments, Antibody 3 and the antibodyvariants (Antibodies 11-15) were more effective than FI6v4 inneutralizing swine-origin A/Indiana/10/2011 H3N2 virus. Antibody 3 andthe antibody variants potently neutralized swine-originA/Minnesota/11/2010 H3N2 virus whereas FI6v4 failed to neutralize at thehighest concentration (50 ug/ml) of antibody tested.

TABLE 6 Neutralizing activity (IC₅₀ ug/ml) Antibody Antibody AntibodyAntibody Antibody Antibody H3N2 virus FI6 v4 3 11 12 13 14 15swine-origin >50 2.2 1.6 1.1 1.6 1.4 0.9 A/Minnesota/11/2010 >50 4.2 1.51.2 1.4 2.3 2.7 swine-origin 13.7 3.1 2.8 2.5 2.2 3.3 5.5A/Indiana/10/2011 29.3 3.7 2.1 1.8 3.9 2.9 3.9

Example 6 Anti-HA Neutralizing Antibody Inhibits Influenza Fusion andProtease-Mediated HA0 Cleavage

To test for the antibody mediated fusion inhibition, a low pH inducedred blood cell fusion assay was performed through a modified protocoldescribed previously (Wang T. T. et al., 2010 PLoS Pathog. 6). In brief,A/Puerto Rico/8/34 virus (10×10⁶ TCID50) was incubated with human redblood cells (2% final red cell concentration) on ice for 10 minutes.Dilutions of Antibody 3, Antibody 12, and a non-relevant antibody MPE8v3were incubated with virus for 30 minutes at RT. The red blood cells werethen added to the virus-antibody mixture for 30 minutes at 37° C. andfinally sodium acetate buffer (0.5 M pH 5.0) was added for additional 45minutes at 37° C. Samples were centrifuged for 6 minutes at 400×g andincubated for additional 45 minutes at RT and then centrifuged again for6 minutes at 400×g to pellet red blood cells. Supernatants were thentransferred to an ELISA plate to determine the amount of released NADPHby measuring absorbance at 540 nm (FIG. 1B). The result showed thatAntibody 3 and Antibody 12 potently inhibited viral fusion whereas theMPE8v3, a human monoclonal antibody against the fusion protein of aparamyxovirus (Corti et al., 2013 Nature 501), was not able to inhibitthe low pH induced fusion.

To test for antibody mediated blockade of the HA maturation, recombinantHA of A/New Caledonia/20/99 (H1N1) was incubated for 40 minutes withAntibody 3, FI6v4, FE17.23 or an isotope control antibody at molar ratioof 15:1 (mAb:HA). The antibody-HA mixture was then exposed to 2.5 ug/mlof TPCK-treated trypsin and incubated for 5, 10 and 20 minutes at 37° C.The samples were separated on a polyacrylamide gel and then transferredto nitrocellulose membrane for Western blot analysis using abiotinylated human mAb (FO32) (Humabs) that recognizes HA2 and HA0 ofinfluenza A strains (FIG. 1C). The result showed that Antibody 3 wasmore potent than FI6v4 in blocking the protease-mediated HA0 cleavage.In contrast, FE17.23, a human monoclonal antibody that recognizes the HAglobular head and control antibody were not able to inhibitprotease-mediated HA0 cleavage. In a separate experiment we compared theprotease cleavage inhibition of Antibody 12 and Antibody 14 incomparison to Antibody 3 using the same conditions described above (FIG.1D). The results showed that Antibody 12, Antibody 13, had a similarability to block the protease cleavage as Antibody 3.

Example 7 Anti-HA Antibodies Exhibit Fc-Effector Function

Antibodies have the potential to clear virus infected cells throughFc-effector function such as antibody dependent cellular cytotoxicity(ADCC), antibody dependent cellular phagocytosis (ADCP), and complementdependent killing (CDC). To confirm the anti-HA antibodies exhibitedADCC activity; we tested their ability to kill virus infected cells inthe presence of human natural killer (NK) cells. The ADCC assay wasperformed on MDCK cells infected with A/Hong Kong/8/68 at an MOI of 20.Infected cells were incubated with a dilution series of antibody, andthen incubated with purified NK cells that were negatively selected fromhuman PBMC (Miltenyi), at an effector to target ratio of 6:1. Theinfected cells, antibody, and NK cells mixtures were incubated for 4hours, and cell killing was measured by LDH release (Roche). FIG. 2shows that all four anti-HA stalk antibodies exhibited dose dependentkilling of infected MDCK cells.

To measure the ability of the anti-HA antibodies to mediatephagocytosis, we used MDCK cells stably transfected with the HA derivedfrom A/Hong Kong/8/68 as target cells. Human monocytes were isolatedfrom PBMCs, and cultured for 7 days in the presence of M-CSF todifferentiate into macrophages. The human macrophages and HA-expressingtarget cells were fluorescently labelled violet and green, respectively(CellTrace Violet or CSFE, Invitrogen). Labelled effector and targetcells were incubated at a 6:1 ratio in the presence of a dilution seriesof antibody for 2 hours, and then analyzed by flow cytometry. Thepercent phagocytosis was measured as the percent ofviolet stainedmacrophages that also were positive for the green target cells (doublepositive). FIG. 3 shows that all the anti-HA antibodies showed similarlevels of ADCP, as expected the nonspecific control antibody showed nophagocytosis.

To measure the ability of the anti-HA antibodies to work with complementto mediate the killing of infected cells, we performed CDC assay. Inthis assay, MDCK cells were infected with A/Puerto Rico/8/34 at an MOIof 2, incubated with a dilution series of antibody, and complementderived from a rabbit (Cedarlane) at an effector to target ratio of1:18. Cell killing was measured by LDH release (Roche). FIG. 4 showsthat all the anti-HA antibodies showed the ability to mediate cellkilling in the presence of complement.

Example 8 Prophylactic and Therapeutic Effect of Anti-HA Antibodies

The protective efficacy of human neutralizing antibody (nAbs) againstinfluenza virus infection was evaluated in six-to-eight weeks' oldBALB/c (Harlan Laboratories) mouse model. Mice were treated withdifferent doses of nAb either before or after lethal viral challenge.

Prophylactic activity (FIGS. 5 & 6) Mice in groups of 8 wereadministered with Antibody 3 as a single intraperitoneal injection (IP)at doses of 0.1, 0.3, 1, 3 and 10 mg/kg, or with a human isotypenon-relevant control IgG at 10 mg/kg in 100 μl volumes. Four hours afterdosing, mice were inoculated intranasally with 7 times the fifty percentmouse lethal dose (7 MLD₅₀) of A/California/7/09 (H1N1) (H1-CA09) or 7:1A/PR/8:A/HK/8/68 HA (H3N1) (H3-HK68) reassortant in a 50 μl volume. Micewere weighed on the day or one day before virus challenge and monitoreddaily for 14 days for weight loss and survival (mice with body weightloss≥25% were euthanized). Antibody 3 conferred protection in adose-dependent manner. IP injection of 1 mg/kg or greater of Antibody 3provided complete protection in animals challenged with H1-CA09 (FIG. 5)and H3-HK68 (FIG. 6). A lower antibody dose (0.3 mg/kg) was also highlyprotective with 90% protection. As expected, none of the mice thatreceived the isotype control mAb at 10 mg/kg survived lethal challengeof infection.Therapeutic activity (FIGS. 7 & 8) Mice were inoculated with 3 MLD₅₀ ofH1-CA09 and injected with Antibody 3 at 24 and 48 hours post infection(h.p.i.) (FIG. 7) or with 5 MLD₅₀ of H3-HK68 at 72, 96 and 120 h.p.i.(FIG. 8). IP treatment with 30 mg/kg of Antibody 3 at 24 and 48 h.p.iprotected 75-100% of mice challenged with H1-CA09, and at 72 and 96h.p.i protected 87.5-100% of mice challenged with H3-HK68. Treatmentwith same dose of non-relevant isotype control antibody at 0 or 24 h.p.iin H1 and H3 models failed to protect mice from lethal challenge with asurvival rate of 0 or 12.5%, respectively.Therapeutic activity of Antibody 3 variants (FIGS. 9 & 10) Mice wereinoculated with 3 MLD₅₀ of H1-CA09 and injected with antibodies 24h.p.i. (FIG. 9) or inoculated with 7 MLD₅₀ H3-HK68 and injected withantibodies 48 h.p.i. (FIG. 10). IP treatment with 2 mg/kg of Antibody 3and variant mAbs (Antibody 11, Antibody 12, and Antibody 14) protected87.5-100% of mice challenged with H1-CA09, and 3 mg/kg dose of thedifferent nAbs protected 50-87.5% of mice challenged with H3-HK68 Asexpected, treatment with same dose of non-relevant isotype controlantibody at 24 or 48 h.p.i in H1 and H3 models failed to protect micewith a survival rate of 0 or 12.5%, respectively.

Example 9 Therapeutic Effect of Anti-HA Antibodies and Small MoleculeInhibitor Oseltamivir

To directly compare the protective efficacy of anti-HA nAbs to smallmolecule neuraminidase (NA) inhibitor, oseltamivir, and the effect ofcombination therapy, we used the influenza murine model of infectiondescribed in Example 8.

Therapeutic Comparison of Anti-HA nAbs and Oseltamivir (FIGS. 11 & 12)Mice were inoculated with 3 MLD₅₀ of H1-CA09 and treated with 10 mg/kgof Antibody 12 or 25 mg/kg BID for 5 days of oseltamivir initiatedeither at 4 hrs prior, 1 day, or 2 days post infection (FIG. 11).Treatment with Antibody 12 prior to and 1 day post infection protected100% of mice challenged with H1-CA09, whereas all animals treated withoseltamivir succumbed to the infection. All animals treated with thesame dose of non-relevant isotype control 4 hours prior to infectiondied with a survival rate of 0%. Additionally, mice were inoculated with7 MLD₅₀ of H3-HK68 then treated with 10 mg/kg of Antibody 12 or 25 mg/kgBID for 5 days of oseltamivir initiated either at 1, 2, 3, or 4 dayspost infection (FIG. 12). Animals treated with Antibody 12 at 1, 2, or 3days post infection showed a survival rate of 100%, whereas treatmentwith oseltamivir at these same time points showed only a 60%-20%survival rate. As expected, mice treated with same dose of non-relevantisotype control antibody 1 day post infection succumbed to the infectionwith a survival rate of 10%.Therapeutic combination of anti-HA nAbs and oseltamivir (FIG. 13) Toassess the additive effect of the combination of anti-HA mAb withoseltamivir, mice were inoculated with 7 MLD₅₀ of H3-HK68 and treatedwith a suboptimal concentration of Antibody 12 (2.5 or 0.3 mg/kg),oseltamivir at 25 mg/kg BID for 5 days, or a combination of Antibody 12(2.5 or 0.3 mg/kg) and oseltamivir at 25 mg/kg BID for 5 days, at day 3post infection (FIG. 13). Treatment with either Antibody 12 oroseltamivir alone protected only 10-20% of the animals whereas treatmentwith the 2.5 mg/kg of Antibody 12 in combination with oseltamivirprotected 80%, and 0.3 mg/kg of Antibody 12 in combination withoseltamivir protected 50% of the animals.

Example 10 Therapeutic Effect of Anti-HA Antibodies and Small MoleculeInhibitor Against H5N1 Influenza Infection in the Ferret

The protective efficacy of anti-HA nAbs and oseltamivir against a highlypathogenic influenza virus infection was evaluated in five-to-sixmonths' old influenza sero-negative ferrets (Triple F Farms). Allferrets were challenged intranasally with 1 LD₉₀ of A/VN/1203/04 (H5N1)highly pathogenic avian influenza virus in 1.0 mL (approximately 0.5mL/nare), and then treated with either a single dose of Antibody 12 at25 mg/kg or oseltamivir at 25 mg/kg BID for 5 days initiated at 1, 2, or3 days post infection. Percent survival was calculated for each group(n=7) (FIG. 14). Ferrets treated with Antibody 12 initiated at 1, 2, and3 days post infection, as well as those treated with oseltamivir 1 daypost infection were protected, having a 100% survival rate. However,when oseltamivir treatment was initiated at 2 and 3 days post infection,ferrets only had 71% survival (mean day of death of 12) and 29% survival(mean day of death 9), respectively. As expected animals treated with 25mg/kg of a non-relevant isotype control antibody at 1 day post infectionfailed to live with a 0% survival rate.

Example 11 Epitope Identification by Selection of Monoclonal AntibodyResistant Mutants (MARMs)

Antibody resistant mutants were isolated using two different methodsfrom three H3N2 viruses. A/Aichi/2/68 (Aichi/68) H3N2 was incubated withhigh concentrations of Antibody 12 (125×IC₅₀) for 1 hour before themixture of virus and antibody was adsorbed to MDCK cells at 30,000TCID50 per well in 10×96-well plates and cultured in the presence ofAntibody 12 (10×IC₅₀). 3 putative Antibody12 HK2/68 MARMs exhibiting thecytopathic effect (CPE) on the infected cells up to 3 days afterinfection were isolated. The HA gene were amplified by RT-PCR andsubsequently sequenced. Sequence analysis revealed 2 nonsynonymoussubstitutions compared with the parental sequence (Table 7). These twonucleotide changes respectively code for single amino acid substitutionsfrom isoleucine (I) to arginine (R); and from aspartic acid (D) totyrosine (Y) at amino acid position 18 and 19 in the highly conservedstalk region of HA2. Alternatively, serial passage of influenza H3N2viruses, A/Wisconsin/67/2005 (W105), and ca A/Panama/2007/1999 (Pa99)were propagated in the presence of increasing concentrations of Antibody12 from 2-5×IC₅₀ up to 100×IC₅₀. Potential escape mutants were subclonedby limited dilution and their cognate HA genes were subjected tosequence analysis. The single amino acid changes from D to Y at position19 and from Glutamine (Q) to R at position 42 in HA2 was identified. Inaddition, double mutations were observed with amino acid substitutionfrom Histine (H) to Q at position 156 in HA1 in combination with D19Y,or from D to asparagine (N) at position 19 in combination with aminoacid change from 1 to N at residue 45 in HA2; or from alanine (A) tothreonine (T) at position 196 in HA1 in combination with Q42R (Table 7).Similarly, when Pa99 was serially passaged in the presence of Antibody12 concentrations up to 100×IC₅₀, single amino acid substitution wasselected at HA2 residue 42 (Q42R) and 45 (145T) (Table 7). Therepresentative MARM variants shown in Table 7 were used in amicroneutralization assay to further evaluate the phenotypicsusceptibility of these MARMs to neutralization by Antibody 12. Theresults showed that the in vitro-selected WI05 MARMs containingmutations D19Y, H156Q/D19Y, D19N/145N, Q42R or A196T/Q42R; Pa99 MARMscontaining Q42R or 145T, and Aichi/68 MARMs harboring mutations D19Y or118R were less susceptible to antibody neutralization, with increases incalculated IC₅₀ values ranging from >8-fold for Pa99 resistant clonesto >180-fold for W105 resistant variants when compared with theirparental wild type strains, respectively (Table 8). To assess the effectof these amino acid substitutions on the susceptibility toneutralization by Antibody 12, recombinant A/Hong Kong/1-5/68 (rHK68) H3variants encoding individual mutations were generated and evaluatedusing a microneutralization assay. As shown in Table 9, the H3rHK68_I18R and rHK68_D19Y variants exhibited resistance to Antibody 12at the highest concentration tested (˜200 μg/mL) and conferred >130-foldreduction in susceptibility to Antibody 12 neutralization compared withwild type rHK68 virus. The single amino acid changes Q42R in rHK68resulted in modest about 8-fold reductions in susceptibility toneutralization by Antibody 12. However, amino acid substitutions (K156Q,A196T, I45N or I45T) identified in the HA proteins of selected MARMs didnot alter the susceptibility of recombinant HK68 viruses encoding suchsubstitutions to Antibody 12 in microneutralization assay. These resultssuggest that Antibody 12 recognizes conformational epitopes in a highlyconserved stalk region of HA2 and amino acids at positions 18, 19 42 or45 are key contact residues.

TABLE 7 Amino acid substitutions identified in the H3 HA of Antibody 12resistant mutants Location in Nucleotide Amino acid HA H3N2 Virus changechange in HA subunits A/Wisconsin/67/2005 G1090T D19Y HA2 C156A, G1090TH156Q, D19Y HA1, HA2 A1160G Q42R HA2 G634A, A1160G A196T, Q42R HA1, HA2G1090A, T1169A D19N, I45N HA2, HA2 ca A/Panama/2007/99 A1160G Q42R HA2T1169C I45T HA2 A/Aichi/2/68 G1090T D19Y HA2 T1088G I18R HA2

TABLE 8 Susceptibility of H3 resistant variants to Antibody 12neutralization (Neut) Amino acid Fold changes changes in HA of Avg.Neut. relative to wild Parental H3N2 virus MARMs tested (μg/ml) typevirus A/Wisconsin/67/2005 wild type 1.09 D19Y >200 >180 Q42R >200 >180H156Q/D19Y >200 >180 D19N/I45N >200 >180 A196T/Q42R >200 >180 caA/Panama/2007/99 wild type 6.68 Q42R >600 >90 I45T 54.51 8.16A/Aichi/2/68 wild type 3.98 D19Y >50 >12 I18R >50 >12

TABLE 9 Susceptibility of rHK68 H3 variants to Antibody 12Neutralization (Neut) Fold changes reassortant Avg. Neut. relative towild type virus_mutation (μg/ml) virus rHK68 wild type 1.42 1rHK68_I18R >200 >130 rHK68_D19N 3.04 2.01 rHK68_D19Y >200 >130rHK68_Q42R 11.13 7.82 rHK68_I45N 1.94 1.28 rHK68_I45T 3.38 2.23rHK68_K156Q 3.33 2.34 rHK68_A196T 4.06 2.85

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Sequence Listing Information Antibody 1 (original cDNA) SEQ ID NO: 1cagatacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcgaataaccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggctgtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggccaagggacaatggtcaccgtctcttcag SEQ ID NO: 2QIQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSS HCDR1 SEQ ID NO: 3 SNNAVWN HCDR2SEQ ID NO: 4 RTYYRSKWYNDYAESVKS HCDR3 SEQ ID NO: 5 SGHITVFGVNVDAFDMSEQ ID NO: 6 gacatccagatcacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccggacaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagggaccaaggtg gaaatcaaaSEQ ID NO: 7 DIQITQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1SEQ ID NO: 8 RTSQSLSSYLH LCDR2 SEQ ID NO: 9 AASSLQS LCDR3 SEQ ID NO: 10QQSRT Antibody 2 (expressed form of Antibody 1) SEQ ID NO: 11caggtacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcgaataaccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggctgtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggccaagggacaatggtcaccgtctcttcag SEQ ID NO: 12QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSS HCDR1 SEQ ID NO: 13 SNNAVWN HCDR2SEQ ID NO: 14 RTYYRSKWYNDYAESVKS HCDR3 SEQ ID NO: 15 SGHITVFGVNVDAFDMSEQ ID NO: 16 gacatccagatgacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccggacaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagggaccaaggtg gaaatcaaaSEQ ID NO: 17 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1SEQ ID NO: 18 RTSQSLSSYLH LCDR2  SEQ ID NO: 19 AASSLQS LCDR3 SEQ ID NO: 20 QQSRT Antibody 3 (codon optimized Antibody 2)SEQ ID NO: 21 caggtccagctgcaggagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactgtcaaccccgatacctccaagaatcagttctctctgcacctgaaaagtgtgacccctgaggacacagccgtgttctactgcgtcagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc SEQ ID NO: 22QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSS HCDR1  SEQ ID NO: 23 SNNAVWN HCDR2 SEQ ID NO: 24 RTYYRSKWYNDYAESVKS HCDR3  SEQ ID NO: 25 SGHITVFGVNVDAFDMSEQ ID NO: 26 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccgactttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtg gagatcaaaSEQ ID NO: 27 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1SEQ ID NO: 28 RTSQSLSSYLH LCDR2 SEQ ID NO: 29 AASSLQS LCDR3SEQ ID NO: 30 QQSRT Antibody 4 (original cDNA) degenerate nucleotidein HCDR3, t or a SEQ ID NO: 31caggtccagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcgagtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagttatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctatatattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaaggggcaaaggtcaccgtgtcttcag SEQ ID NO: 32QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWLGRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARGGHITVFGLNIDAYDIWGQGAKVTVSS HCDR1  SEQ ID NO: 33 SNSAVWN HCDR2SEQ ID NO: 34 RTYYRSKWYYDYAESVKS HCDR3 SEQ ID NO: 35 GGHITVFGLNIDAYDISEQ ID NO: 36 gacatccaggtgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccgggcacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctcaccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggaccaaggttgaa atcaaac SEQ ID NO: 37DIQVTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATTLQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVE IK LCDR1SEQ ID NO: 38 RAQSLSSYLH LCDR2 SEQ ID NO: 39 AATTLQS LCDR3 SEQ ID NO: 40QQSRT Antibody 5 (expressed form of Antibody 4 HCDR3 V) SEQ ID NO: 41caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcgagtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagttatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctatatattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaaggggcaatggtcaccgtctcttcag SEQ ID NO: 42QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWLGRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARGGHITVFGLNIDAYDIWGQGAMVTVSS HCDR1 SEQ ID NO: 43 SNSAVWN HCDR2SEQ ID NO: 44 RTYYRSKWYYDYAESVKS HCDR3 SEQ ID NO: 45 GGHITVFGLNIDAYDISEQ ID NO: 46 gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccgggcacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctcaccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggaccaaggtggag atcaaac SEQ ID NO: 47DIQMTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATTLQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVE IK LCDR1SEQ ID NO: 48 RAQSLSSYLH LCDR2 SEQ ID NO: 49 AATTLQS LCDR3 SEQ ID NO: 50QQSRT Antibody 6 (expressed form of Antibody 4 HCDR3 E) SEQ ID NO: 51caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcgagtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagttatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctatatattactgtgcaagaggtggccacattacggagtttgggctgaatattgacgcttatgatatttggggccaaggggcaatggtcaccgtctcttcag SEQ ID NO: 52QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWLGRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARGGHITEFGLNIDAYDIWGQGAMVTVSS HCDR1 SEQ ID NO: 53 SNSAVWN HCDR2 SEQ ID NO: 54 RTYYRSKWYYDYAESVKS HCDR3  SEQ ID NO: 55 GGHITEFGLNIDAYDISEQ ID NO: 56 gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccgggcacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctcaccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggaccaaggtggag atcaaac SEQ ID NO: 57DIQMTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATTLQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVE IK LCDR1 SEQ ID NO: 58 RAQSLSSYLH LCDR2  SEQ ID NO: 59 AATTLQS LCDR3 SEQ ID NO: 60 QQSRT Antibody 7 (original cDNA) SEQ ID NO: 61caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcatctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagtcatcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtgtatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctcgggacaaaggtcaccgtctcttcag SEQ ID NO: 62QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRQSPLRGLEWLGRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCARGGHITVFGVNIDAFDIWGLGTKVTVSS HCDR1 SEQ ID NO: 63 SNRATWN HCDR2SEQ ID NO: 64 RTYYRSKWYNDYAVSVKS HCDR3 SEQ ID NO: 65 GGHITVFGVNIDAFDISEQ ID NO: 66 gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccgggcaagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatctatgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcactctcaccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgggaccaaggtt gaaatcaaacSEQ ID NO: 67 DIQVTQSPSSLSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATSTLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKV EIK LCDR1 SEQ ID NO: 68 RASQRLNSYLH LCDR2  SEQ ID NO: 69 ATSTLQS LCDR3 SEQ ID NO: 70 QLSRT Antibody 8 (expressed form of Antibody 7)SEQ ID NO: 71 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcatctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagtcatcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtgtatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctcgggacaaaggtcaccgtctcttcag SEQ ID NO: 72QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRASPLRGLEWLGRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCARGGHITVFGVNIDAFDIWGLGTKVTVSS HCDR1 SEQ ID NO: 73 SNRATWN HCDR2SEQ ID NO: 74 RTYYRSKWYNDYAVSVKS HCDR3 SEQ ID NO: 75 GGHITVFGVNIDAFDISEQ ID NO: 76 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccgggcaagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatctatgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcactctcaccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgggaccaaggtg gaaatcaaacSEQ ID NO: 77 DIQMTQSPSSLSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATSTLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKV EIK LCDR1 SEQ ID NO: 78 RASQRLNSYLH LCDR2  SEQ ID NO: 79 ATSTLQS LCDR3 SEQ ID NO: 80 QLSRT Antibody 9 (original cDNA) SEQ ID NO: 81caagtagagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataaccatcaatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggctttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggccaagggacaatggccaccgtctcttcag SEQ ID NO: 82QVELQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARGGHITVFGVNIHCDRIDAFDVWGQGTMATVSS HCDR1 SEQ ID NO: 83 SNSATWN HCDR2SEQ ID NO: 84 RTYYRSKWYNDYADFLKR HCDR3 SEQ ID NO: 85 GGHITVFGVNIDAFDVSEQ ID NO: 86 gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccggacaagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatctatgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaagggaccaaggtt gaaatcaaacSEQ ID NO: 87 DIQVTQSPSSLSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKV EIK LCDR1SEQ ID NO: 88 RTSQSLRSYLH LCDR2 SEQ ID NO: 89 ASSTLQS LCDR3SEQ ID NO: 90 QLSRT Antibody 10 (expressed form of Antibody 9)SEQ ID NO: 91 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataaccatcaatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggctttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggccaagggacaatggtcaccgtctcttcag SEQ ID NO: 92QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARGGHITVFGVNIDAFDVWGQGTMVTVSS HCDR1  SEQ ID NO: 93 SNSATWN HCDR2 SEQ ID NO: 94 RTYYRSKWYNDYADFLKR HCDR3  SEQ ID NO: 95 GGHITVFGVNIDAFDVSEQ ID NO: 96 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccggacaagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatctatgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaagggaccaaggtg gagatcaaacSEQ ID NO: 97 DIQMTQSPSSLSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKV EIK LCDR1 SEQ ID NO: 98 RTSQSLRSYLH LCDR2  SEQ ID NO: 99 ASSTLQS LCDR3 SEQ ID NO: 100 QLSRT Antibody 11 SEQ ID NO: 101caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccgaattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc SEQ ID NO: 102QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDHCDRIAFDMWGQGTMVTVSS HCDR1 SEQ ID NO: 103 SYNAVWN HCDR2 SEQ ID NO: 104 RTYYRSGWYNDYAESVKS HCDR3  SEQ ID NO: 105 SGHITVFGVNVDAFDMSEQ ID NO: 106 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgactttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtg gagatcaaaSEQ ID NO: 107 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1 SEQ ID NO: 108 RTSQSLSSYTH LCDR2  SEQ ID NO: 109 AASSRLS LCDR3 SEQ ID NO: 110 QQSRT Antibody 12 SEQ ID NO: 111caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccgaattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc SEQ ID NO: 112QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSS HCDR1  SEQ ID NO: 113 SYNAVWN HCDR2 SEQ ID NO: 114 RTYYRSGWYNDYAESVKS HCDR3  SEQ ID NO: 115 SGHITVFGVNVDAFDMSEQ ID NO: 116 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccgactttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtg gagatcaaaSEQ ID NO: 117 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1 SEQ ID NO: 118 RTSQSLSSYTH LCDR2  SEQ ID NO: 119 AASSRGS LCDR3 SEQ ID NO: 120 QQSRT Antibody 13 SEQ ID NO: 121caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccgaattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc SEQ ID NO: 122QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSS HCDR1  SEQ ID NO: 123 SYNAVWN HCDR2 SEQ ID NO: 124 RTYYRSGWYNDYAESVKS HCDR3  SEQ ID NO: 125 SGHITVFGVNVDAFDMSEQ ID NO: 126 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacgaccactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgactttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatc aaa SEQ ID NO: 127DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYDHWYQQKPGKAPKLLIYAASSRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEI K LCDR1 SEQ ID NO: 128 RTSQSLSSYDH LCDR2  SEQ ID NO: 129 AASSRLS LCDR3 SEQ ID NO: 130 QQSRT Antibody 14 SEQ ID NO: 131caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggaccacagtcaccgtctcctca SEQ ID NO: 132QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTTVTVSS HCDR1  SEQ ID NO: 133 SNNAVWN HCDR2 SEQ ID NO: 134 RTYYRSKWYNDYAESVKS HCDR3  SEQ ID NO: 135 SGHITVFGVNVDAFDMSEQ ID NO: 136 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgactttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtg gagatcaaaSEQ ID NO: 137 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1 SEQ ID NO: 138 RTSQSLSSYTH LCDR2  SEQ ID NO: 139 AASSRLS LCDR3 SEQ ID NO: 140 QQSRT Antibody 15 SEQ ID NO: 141caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggaccacagtcaccgtctcctca SEQ ID NO: 142QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTTVTVSS HCDR1  SEQ ID NO: 143 SNNAVWN HCDR2 SEQ ID NO: 144 RTYYRSKWYNDYAESVKS HCDR3  SEQ ID NO: 145 SGHITVFGVNVDAFDMSEQ ID NO: 146 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagytcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccgactttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtg gagatcaaaSEQ ID NO: 147 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1 SEQ ID NO: 148 RTSQSLSSYTH LCDR2  SEQ ID NO: 149 AASSRGS LCDR3 SEQ ID NO: 150 QQSRT Antibody 3-GL SEQ ID NO: 151caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggaccacagtcaccgtctcctca SEQ ID NO: 152QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTTVTVSS HCDR1  SEQ ID NO: 153 SNNAVWN HCDR2 SEQ ID NO: 154 RTYYRSKWYNDYAESVKS HCDR3  SEQ ID NO: 155 SGHITVFGVNVDAFDMSEQ ID NO: 156 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccgactttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtg gagatcaaaSEQ ID NO: 157 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKV EIK LCDR1 SEQ ID NO: 158 RTSQSLSSYLH LCDR2  SEQ ID NO: 159 AASSLQS LCDR3 SEQ ID NO: 160 QQSRT

The invention claimed is:
 1. An isolated antibody or a binding fragmentthereof that is capable of binding to influenza A virus hemagglutininand neutralizing at least one group 1 subtype and at least 1 group 2subtype of influenza A virus, wherein the antibody or fragment thereofincludes a set of six CDRs which includes HCDR1 of SEQ ID NO.: 113,HCDR2 of SEQ ID NO.: 114, HCDR3 of SEQ ID NO.: 115, LCDR1 of SEQ ID NO.:118, LCDR2 of SEQ ID NO.: 119 and LCDR3 of SEQ ID NO.:
 120. 2. Theantibody or binding fragment according to claim 1, wherein the antibodyor binding fragment is capable of neutralizing one or more influenza Avirus group 1 subtype selected from: H1, H2, H5, H6, H8, H9, H11, H12,H13, H16 and variants thereof; and one or more influenza A virus group 2subtypes selected from: H3, H4, H7, H10, H14 and H15 and variantsthereof.
 3. The antibody or binding fragment thereof according to claim1, wherein the antibody or binding fragment is capable of neutralizinggroup 1 subtypes: H1, H2, H5, H6 and H9 and group 2 subtypes H3 and H7;or wherein the antibody or binding fragment is capable of neutralizinggroup 1 subtypes: H1, H2, H5 and H6 and group 2 subtypes H3 and H7. 4.The antibody or binding fragment thereof according to claim 1, whereinthe antibody or binding fragment has high neutralizing potency expressedas 50% inhibitory concentration (IC₅₀ ug/ml) in the range of from about0.01ug/ml to about 50ug/ml of antibody for neutralization of influenza Avirus in a microneutralization assay.
 5. The antibody or bindingfragment thereof according to claim 1 comprising a VH having at least75% identity and/or a VL having at least 75% identity to a VH of SEQ IDNO.: 112 and a VL of SEQ ID NO.:
 117. 6. The antibody or bindingfragment thereof according to claim 1 comprising a VH of SEQ ID NO.: 112and a VL of SEQ ID NO.:
 117. 7. The antibody or binding fragment thereofaccording to claim 1, wherein the antibody or binding fragment isselected from the group consisting of: an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahumanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linkedFv, a scFv, a single domain antibody, a diabody, a multispecificantibody, a dual-specific antibody, and a bispecific antibody.
 8. Theantibody or binding fragment thereof according to claim 1, wherein theVH comprises human germline framework VH6-1, the VL comprises humangermline framework VK1-39, and combination thereof.
 9. The antibody orbinding fragment thereof according to claim 1 comprising an Fc region.10. The antibody or binding fragment thereof according to claim 1,wherein the antibody is an IgG1, IgG2 or IgG4 or fragment thereof. 11.An isolated nucleic acid encoding an antibody or binding fragmentthereof according to claim
 1. 12. A vector comprising an isolatednucleic acid according to claim
 11. 13. A host cell comprising a nucleicacid according to claim
 11. 14. A composition comprising an antibody orbinding fragment thereof according to claim 1 and a pharmaceuticallyacceptable carrier.
 15. A composition comprising an antibody or bindingfragment thereof according to claim 1 comprising 25mM His and 0.15M NaClat pH 6.0.